Receptors providing targeted costimulation for adoptive cell therapy

ABSTRACT

Some embodiments provided herein are chimeric costimulatory antigen receptor (CoStAR), useful in adoptive cell therapy (ACT), and cells comprising the CoStAR. In some embodiments, the CoStAR can act as a modulator of cellular activity enhancing responses to defined antigens. In some embodiments, CoStAR and/or fusion proteins, nucleic acids encoding the CoStAR and therapeutic uses thereof are also provided.

RELATED APPLICATIONS AND INCORPORATION BY REFERENCE

This application is a continuation of U.S. patent application Ser. No.17/807,109 filed Jun. 15, 2022, which claims the benefit of U.S.Provisional Ser. No. 63/211,042 and U.S. Provisional Ser. No.63/211,046, both filed Jun. 16, 2021; U.S. Provisional Ser. No.63/222,913, filed Jul. 16, 2021; U.S. Provisional Ser. No. 63/301,340filed Jan. 20, 2022, each of which is hereby incorporated by referencein their entireties.

SEQUENCE STATEMENT

The instant application contains a Sequence Listing, which has beensubmitted electronically and is hereby incorporated by reference in itsentirety. The sequence listing, was created on Sep. 22, 2022, is namedSeqListing-INSTB007C1 and is 849,344 bytes in size.

Reference is made to GB patent application Serial No. 1900858.0, filed22 Jan. 2019, U.S. patent application Ser. No. 62/951,770, filed 20 Dec.2019, International application PCT/GB2020/050120, filed 20 Jan. 2020,and U.S. provisional patent applications 63/053,494 and 63/053,498,filed Jul. 17, 2020.

The foregoing applications, and all documents cited therein or duringtheir prosecution (“appln cited documents”) and all documents cited orreferenced in the appln cited documents, and all documents cited orreferenced herein (“herein cited documents”), and all documents cited orreferenced in herein cited documents, together with any manufacturer'sinstructions, descriptions, product specifications, and product sheetsfor any products mentioned herein or in any document incorporated byreference herein, are hereby incorporated herein by reference, and maybe employed in the practice of the invention. More specifically, allreferenced documents are incorporated by reference to the same extent asif each individual document was specifically and individually indicatedto be incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to a chimeric costimulatory antigenreceptor (CoStAR) useful in adoptive cell therapy (ACT), and cellscomprising the CoStAR. The CoStAR can act as a modulator of cellularactivity enhancing responses to defined antigens. The present inventionalso provides CoStAR and/or fusion proteins, nucleic acids encoding theCoStAR and therapeutic uses thereof.

BACKGROUND

Adoptive cell therapy (ACT) using autologous T-cells to mediate cancerregression has shown much promise in early clinical trials. Severalgeneral approaches have been taken such as the use of naturallyoccurring tumor reactive or tumor infiltrating lymphocytes (TILs)expanded ex vivo. Additionally, T-cells may be genetically modified toretarget them towards defined tumor antigens. This can be done via thegene transfer of peptide (p)-major histocompatibility complex (MHC)specific T-cell Receptors (TCRs) or synthetic fusions between tumorspecific single chain antibody fragment (scFv) and T-cell signalingdomains (e.g. CD3ζ), the latter being termed chimeric antigen receptors(CARs).

TIL and TCR transfer has proven particularly good when targetingmelanoma (Rosenberg et al. 2011; Morgan 2006), whereas CAR therapy hasshown much promise in the treatment of certain B-cell malignancies(Grupp et al. 2013).

Costimulatory signals are useful to achieve robust CAR T cell expansion,function, persistence and antitumor activity. The success of CAR therapyin leukemia has been partly attributed to the incorporation ofcostimulatory domains (e.g. CD28 or CD137) into the CAR construct,signals from which synergize with the signal provided by CD3ζ to enhanceanti-tumor activity. The basis of this observation relates to theclassical signal 1/signal 2 paradigm of T-cell activation. Here signal1, provided by the TCR complex, synergizes with signal 2 provided bycostimulatory receptors such as CD28, CD137 or CD134 to permit the cellsto undergo clonal expansion, IL2 production and long term survivalwithout the activation induced cell death (AICD) associated with signal1 alone. Furthermore the involvement of signal 2 enhances the signalgenerated through signal 1 allowing the cells to respond better to lowavidity interactions such as those encountered during anti-tumorresponses.

Citation or identification of any document in this application is not anadmission that such document is available as prior art to the presentinvention.

SUMMARY

Provided herein are novel chimeric costimulatory antigen receptors(CoStARs) that bind to carcinoembryonic antigen (CEA) and/or mesothelin(MSLN) and cells comprising or expressing the CoStARs which arebeneficial for CAR and non-CAR based T-cell therapies alike. Providedherein are cells that express a novel chimeric costimulatory receptor toprovide a costimulatory signal to T-cells upon engagement with a defineddisease-associated, for example tumor-associated, antigen.

There have been several reports in which split signal 1 and signal 2have been used to drive antigen specific responses in engineered T-cells(Alvarez-Vallina & Hawkins 1996). However, none have utilized the fulllength CD28 molecule. There are specific advantages to using full lengthreceptors, such as CD28 as opposed to truncated forms. A full lengthreceptors may be capable of dimerization, enabling the receptor tofunction in its native form, indeed chimeric antigen receptors fail tofunction optimally when expressed as a monomer (Bridgeman et al. 2010).

In an embodiment, a CoStAR induces signal 2 upon engagement with adefined antigen such as a disease associated or tumor associatedantigen. The full length CD28 molecule contains motifs critical to itsnative function in binding members of the B7 family of receptors;although this is potentially dangerous from the perspective of CARscarrying CD28 and CD3ζ receptors in tandem, wherein ligation of CAR byB7 could trigger T-cell activation, there are beneficial qualities forreceptors harbouring signal 2 receptors alone. Provided herein is atargeted chimeric costimulatory receptor (CoStAR) which comprises anextracellular binding domain operatively linked to a transmembranedomain, a first signaling domain, and a CD40 signaling domain or asignaling fragment thereof. It has been discovered that costimulatoryreceptors comprising a CD40 signaling domain display novel and improvedactivity profiles.

In some embodiments, the CD40 signaling domain comprises SEQ ID NO:32,SEQ ID NO:33, or SEQ ID NO:34. In some embodiments, the CD40 signalingfragment comprises an SH3 motif (KPTNKAPH, SEQ ID NO:35), TRAF2 motif(PKQE, SEQ ID NO:36, PVQE, SEQ ID NO:37, SVQE, SEQ ID NO:38), TRAF6motif (QEPQEINFP, SEQ ID NO:39), PKA motif (KKPTNKA, SEQ ID NO:40,SRISVQE, SEQ ID NO:41), or a combination thereof, or is a full lengthCD40 intracellular domain. In some embodiments, one or more of the SH3,TRAF2, TRAF6, or PKA motifs of the CD40 signaling domain is mutated.

In some embodiments, the first signaling domain of the CoStAR comprisesa signaling domain or signaling fragment of a receptor, such as, forexample a tumor necrosis factor receptor superfamily (TNFRSF) receptor,including but not limited to CD2, CD9, CD26, CD27, CD28, CD29, CD38,CD40, CD43, CD46, CD49d, CD55, CD73, CD81, CD82, CD99, CD100, CD134(OX40), CD137 (41BB), CD150 (SLAM), CD270 (HVEM), CD278 (ICOS), DAP10,NTKR, CD357 (GITR), or EphB6. In some embodiments, the CoStAR comprisesCD2, CD9, CD26, CD27, CD28, CD29, CD38, CD40, CD43, CD46, CD49d, CD55,CD73, CD81, CD82, CD99, CD100, CD134 (OX40), CD137 (41BB), CD150 (SLAM),CD270 (HVEM), CD278 (ICOS), DAP10, NTKR, CD357 (GITR), or EphB6. Inembodiments, wherein the first signaling domain comprises a CD40signaling domain thus the CoStAR comprises elements of two CD40signaling domains.

In some embodiments, the CoStAR comprises a second signaling domain orsignaling fragment of a receptor, such as, for example a tumor necrosisfactor receptor superfamily (TNFRSF) receptor, including but not limitedto CD2, CD9, CD26, CD27, CD28, CD29, CD38, CD40, CD43, CD46, CD49d,CD55, CD73, CD81, CD82, CD99, CD100, CD134 (OX40), CD137 (41BB), CD150(SLAM), CD270 (HVEM), CD278 (ICOS), CD357 (GITR), or EphB6. The firstsignaling domain or signaling fragment, the CD40 signaling domain orsignaling fragment, and the second signaling domain or signalingfragment can be in any order. Exemplary embodiments include, withoutlimitation, CoStAR which comprise CD28, CD137, and CD40 signalingdomains, CD28, CD134, and CD40 signaling domains, CD28, CD2, and CD40signaling domains, CD28, GITR, and CD40 signaling domains, CD28, CD29,and CD40 signaling domains, or CD28, CD150, and CD40 signaling domains.

In some embodiments, the extracellular antigen-binding domain (e.g.,without limitation CEA-binding domain, MSLN-binding domain) of a CoStARof the invention is operatively linked to the transmembrane domain by alinker and/or a spacer. In some embodiments, the linker comprises fromabout 5 to about 20 amino acids. In some embodiments, the linkercomprises AAAGSGGSG (SEQ ID NO:18).

In some embodiments, a CoStAR comprises a spacer which operatively linksthe extracellular binding domain to the transmembrane domain andcomprises from about 10 to about 250 amino acids. In some embodiments,the spacer comprises an extracellular sequence of CD8 or CD28 or afragment thereof. In some embodiments, the CoStAR comprises a secondextracellular binding domain. In some embodiments, the second bindingdomain comprises an extracellular ligand binding domain from CD8 orCD28. In some embodiments, the spacer comprises one or moreimmunoglobulin domains or an immunoglobulin constant region. In someembodiments, the spacer comprises one or more immunoglobulin domains oran immunoglobulin constant region such as, without limitation, SEQ IDNO:24.

In some embodiments the transmembrane domain of a CoStAR comprises atransmembrane domain of a TNFRSF protein. In some embodiments, atransmembrane domain of a CoStAR comprises a transmembrane domain ofCD28 or CD8. In some embodiments, a transmembrane domain of a CoStARcomprises a transmembrane sequence of CD28 or CD8.

In some embodiments, the CoStARs are useful to stimulate an immuneresponse against a selected target that expresses a tumor associatedantigen (TAA), e.g. without limitation, carcinoembryonic antigen (CEA),mesothelin (MSLN), or other. In some embodiments, the CoStAR comprisesan antigen binding fragment of the scFv of SEQ ID NO:9, SEQ ID NO:10,SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, or SEQ ID NO:14, e.g., afragment comprising one, two, three, four, five, or all sixcomplementary determining regions (CDRs). In some embodiments, theCoStAR comprises the scFv of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11,SEQ ID NO:12, SEQ ID NO:13, or SEQ ID NO:14. In some embodiments, theCoStAR comprises an antigen binding fragment of the scFv of a) SEQ IDNO:186, SEQ ID NO:187, SEQ ID NO:188, SEQ ID NO:189, SEQ ID NO:190, orSEQ ID NO:191; or b) SEQ ID NO:510, SEQ ID NO:511, SEQ ID NO:512, or SEQID NO:513, e.g., a fragment comprising one, two, three, four, five, orall six CDRs. In some embodiments, the CoStAR comprises the scFv of a)SEQ ID NO:186, SEQ ID NO:187, SEQ ID NO:188, SEQ ID NO:189, SEQ IDNO:190, SEQ ID NO:191; or b) SEQ ID NO:508, SEQ ID NO:509, SEQ IDNO:510, SEQ ID NO:511, SEQ ID NO:512, or SEQ ID NO:513.

In some embodiments, an extracellular binding domain can comprise,without limit, an scFv, a peptide, an antigen binding portion of anantibody, an antibody heavy-chain variable domain, an antibody lightchain variable domain, a single domain antibody, a CEA ligand, or anMSLN ligand.

In some embodiments, a CoStAR comprises a CD3ζ signaling domain, forexample located at the C-terminus.

In some embodiments, a CoStAR comprises an N-terminal signal peptide. Insome embodiments, the N-terminal signal peptides are signal peptides ofoncostatin M (OSM), CD8a, CD2, interleukin-2 (IL-2),granulocyte-macrophage colony stimulating factor (GM-CSF), and humanIgGκ.

In some embodiments, there is provided a nucleic acid which encodes aCoStAR. The nucleic acid may be optimized, for example be codonoptimized for expression in a host cell. In some embodiments, thenucleic acid is codon optimized for expression in a human cell.

In some embodiments, there is provided vector which encodes and iscapable of expressing a CoStAR.

In some embodiments, there is provided a cell which expresses a CoStAR.In some embodiments, the cell expresses two or more CoStARs, for examplethe cell expresses a CoStAR that binds to CEA or MSLN and a CoStAR thatbinds to FOLR1 or a CoStAR that binds to CA125, such as but not limitedto anti-CEA.CD28.CD40 and anti-CA125.41BB.CD40 or anti-MSLN.CD28.CD40and anti-CA125.41BB.CD40. In some embodiments, the cell expresses aCoStAR which binds to CEA and a CoStAR which binds to PDL1, such as butnot limited to anti-CEA.CD28.CD40 and PD1.CD28.CD40 or expresses aCoStAR which binds to MSLN and a CoStAR which binds to PDL1, such as butnot limited to anti-MSLN.CD28.CD40 and PD1.CD28.CD40.

In some embodiments, a cell engineered to express a CoStAR comprises analpha-beta T cell, gamma-delta T cell, T regulatory cell, TIL, NKT cellor NK cell. In some embodiments, a cell engineered to express a CoStARcoexpresses a chimeric antigen receptor (CAR) or a T cell receptor(TCR).

In some embodiments, provided herein is a method of making the cellwhich expresses a CoStAR which comprises transducing or transfecting acell with a vector which encodes and is capable of expressing a CoStAR.

In some embodiments, a method is provided for preparing a population ofcells that express a CoStAR by transducing or transfecting cells,detecting expression of the CoStAR and enriching, expanding, and/orselecting cells that express the CoStAR.

In some embodiments, provided herein is a method of treating a diseasein a subject by administering a population of cells which express aCoStAR.

In some embodiments, a chimeric costimulatory antigen receptor (CoStAR)is provided which comprises: an extracellular binding domain that bindsto carcinoembryonic antigen (CEA), or an extracellular binding domainthat binds to mesothelin (MSLN), operatively linked to a transmembranedomain, and a first signaling domain and an intracellular domain of ICOSor a signaling fragment thereof, or a first signaling domain and anintracellular domain of NTRK1 or a signaling fragment thereof, or afirst signaling domain and an intracellular domain of DAP10 or asignaling fragment thereof, or a first signaling domain and a CD40signaling domain or a signaling fragment thereof, or a first signalingdomain and one or more of a TRAF2/TRAF3 sequence, a TRAF6 sequence, aTRAF2 sequence, or an IProx sequence.

In some embodiments, a CoStAR is provided where the first signalingdomain comprises a signaling domain or signaling fragment of CD2, CD9,CD26, CD27, CD28, CD29, CD38, CD40, CD43, CD46, CD49d, CD55, CD73, CD81,CD82, CD99, CD100, CD134 (OX40), CD137 (41BB), CD150 (SLAM), CD270(HVEM), CD278 (ICOS), CD357 (GITR), or EphB6.

In some embodiments, a CoStAR is provided where the CoStAR comprises asecond signaling domain.

In some embodiments, a CoStAR is provided where the second signalingdomain comprises a signaling domain or signaling fragment of CD2, CD9,CD26, CD27, CD28, CD29, CD38, CD40, CD43, CD46, CD49d, CD55, CD73, CD81,CD82, CD99, CD100, CD134 (OX40), CD137 (41BB), CD150 (SLAM), CD270(HVEM), CD278 (ICOS), CD357 (GITR), or EphB6.

In some embodiments, a CoStAR is provided where the CD40 signalingfragment comprises an SH3 motif (KPTNKAPH, SEQ ID NO:35), TRAF2 motif(PKQE, SEQ ID NO:36, PVQE, SEQ ID NO:37, SVQE, SEQ ID NO:38), TRAF6motif (QEPQEINFP, SEQ ID NO:39), PKA motif (KKPTNKA, SEQ ID NO:40,SRISVQE, SEQ ID NO:41), or a combination thereof, or is a full lengthCD40 intracellular domain.

In some embodiments, a CoStAR is provided where the first signalingdomain comprises a full length costimulatory domain.

In some embodiments, a CoStAR is provided where the extracellularbinding domain is operatively linked to the transmembrane domain by alinker and/or a spacer.

In some embodiments, a CoStAR is provided where the linker comprisesfrom about 5 to about 20 amino acids.

In some embodiments, a CoStAR is provided where the linker or spacercomprises from about 10 to about 250 amino acids.

In some embodiments, a CoStAR is provided where the CoStAR comprises asecond extracellular binding domain.

In some embodiments, a CoStAR is provided where the second extracellularbinding domain comprises a ligand binding domain from CD8, CD28, orICOS.

In some embodiments, a CoStAR is provided where the transmembrane domaincomprises a transmembrane domain from CD28, CD8, ICOS, DAP10, or NTRK.

In some embodiments, a CoStAR is provided where the transmembrane domaincomprises the transmembrane domain sequence of SEQ ID NO:20, SEQ IDNO:21, or SEQ ID NO:22.

In some embodiments, a CoStAR is provided where the extracellularbinding domain comprises an scFv, a peptide, an antibody heavy-chainvariable domain, an antibody light-chain variable domain, or a CEAligand or a MSLN ligand.

In some embodiments, a CoStAR is provided which further comprises asignaling domain at the C-terminus.

In some embodiments, a CoStAR is provided which further comprises anN-terminal signal peptide.

In some embodiments, a CoStAR is provided where the N-terminal signalpeptide comprises the signal peptide of oncostatin M (OSM), CD8a, CD2,interleukin-2 (IL-2), granulocyte-macrophage colony stimulating factor(GM-CSF), or human IgGκ.

In some embodiments, a nucleic acid which encodes any of the CoStARconstructs described above is provided.

In some embodiments, a CoStAR is provided where a vector comprises theCoStAR construct nucleic acid.

In some embodiments, a cell which expresses any of the CoStARs describedabove is provided.

In some embodiments, the CoStAR expressing cell comprises an alpha-betaT cell, gamma-delta T cell, T regulatory cell, TIL, NKT cell or NK cell.

In some embodiments, the CoStAR expressing cell coexpresses a CAR or aTCR.

In some embodiments, a method of making the CoStar expressing cell isprovided. In some embodiments, the method comprises the step oftransducing or transfecting a cell with a vector.

In some embodiments, a method for preparing a population of cells thatexpress a CoStAR of any one of the CoStARs described above is provided.In some embodiments, the method comprises i) detecting expression of theCoStAR on the surface of cells transfected or transduced with a vectorof claim 19; and ii) selecting cells which are identified as expressingthe CoStAR.

In some embodiments, a cell population is provided which is enriched forcell expression of a of any one of the CoStAR constructs provided above.

In some embodiments, a method for treating a disease in a subject isprovided. In some embodiments the method comprises the step ofadministering a CoStAR expressing cell, or a CoStAr enriched cellpopulation to the subject.

In some embodiments, a fusion protein is provided. In some embodiments,the fusion protein comprises: a binding domain specific for CEA linkedto; a transmembrane domain that is linked to; an ICOS domain that islinked to; a CD40 signaling domain.

In some embodiments, a fusion protein is provided. In some embodiments,the fusion protein comprises: a binding domain specific for MSLN linkedto; a transmembrane domain that is linked to; a CD28 domain that islinked to; a CD40 signaling domain.

In some embodiments, a fusion protein is provided. In some embodiments,the fusion protein comprises: a first sequence that is at least 70%identical to SEQ ID NO: 12; a second sequence that is a transmembranedomain; a third sequence that is at least 70% identical to SEQ ID NO:518; and a fourth sequence that is at least 70% identical to SEQ ID NO:32.

In some embodiments, a fusion protein is provided. In some embodiments,the fusion protein comprises: a first sequence that is at least 70%identical to any one of SEQ ID NO: 186-191; a second sequence that is atransmembrane domain; a third sequence that is at least 70% identical toSEQ ID NO: 25; and a fourth sequence that is at least 70% identical toSEQ ID NO: 32.

In some embodiments, a fusion protein is provided. In some embodiments,the fusion protein comprises: a HCDR1 that is an HCDR1 in SEQ ID NO: 12;a HCDR2 that is an HCDR2 in SEQ ID NO: 12; a HCDR3 that is an HCDR3 inSEQ ID NO: 12; a LCDR1 that is an LCDR1 in SEQ ID NO: 12; a LCDR2 thatis an LCDR2 in SEQ ID NO: 12; a LCDR3 that is an HCDR3 in SEQ ID NO: 12.In some embodiments, 1, 2, 3, 4, 5, or 6 of the LCDRs can include 1, 2,or 3 point mutations. In some embodiments the fusion protein furthercomprises a second sequence that is a transmembrane domain; a thirdsequence that is at least 70% identical to SEQ ID NO: 515; and a fourthsequence that is at least 70% identical to SEQ ID NO: 32.

In some embodiments, the fusion protein further comprises a signalpeptide sequence that is at least 70% identical to SEQ ID NO: 1.

In some embodiments, the fusion protein further comprises a linkersequence that is at least 70% identical to SEQ ID NO: 18.

In some embodiments, the fusion protein further comprises an ICOSsequence that is at least 70% identical to SEQ ID NO: 515.

In some embodiments, the fusion protein further comprises an CD40sequence that is at least 70% identical to SEQ ID NO: 32.

In some embodiments, a fusion protein is provided. In some embodiments,the fusion protein comprises: a HCDR1 that is an HCDR1 in SEQ ID NOs:186-191; a HCDR2 that is an HCDR2 in SEQ ID NOs: 186-191; a HCDR3 thatis an HCDR3 in SEQ ID NOs: 186-191; a LCDR1 that is an LCDR1 in SEQ IDNOs: 186-191; a LCDR2 that is an LCDR2 in SEQ ID NOs: 186-191; a LCDR3that is an HCDR3 in SEQ ID NOs: 186-191. In some embodiments, 1, 2, 3,4, 5, or 6 of the LCDRs can include 1, 2, or 3 point mutations. In someembodiments, the fusion protein further comprises a second sequence thatis a transmembrane domain; a third sequence that is at least 70%identical to SEQ ID NO: 25; and a fourth sequence that is at least 70%identical to SEQ ID NO: 32

In some embodiments, the fusion protein further comprises a signalpeptide sequence that is at least 70% identical to SEQ ID NO: 1.

In some embodiments, the fusion protein further comprises a linkersequence that is at least 70% identical to SEQ ID NO: 18.

In some embodiments, the fusion protein further comprises an CD28 TMsequence that is at least 70% identical to SEQ ID NO: 19.

In some embodiments, the fusion protein further comprises an CD28sequence that is at least 70% identical to SEQ ID NO: 25.

In some embodiments, the fusion protein further comprises an CD40sequence that is at least 70% identical to SEQ ID NO: 32.

In some embodiments, a method of cell therapy is provided comprising: a)identifying a subject, wherein the subject has cancer that expressesMSLN or CEA; and b) administering any one or more of the CoSTaRs orfusion proteins described above.

In some embodiments, a method of treating a cancer in a subject thatexpresses MSLN or CEA is provided, the method comprising: a) identifyinga subject, wherein the subject has cancer that expresses MSLN or CEA;and b) administering any one or more of the CoSTaRs or fusion proteinsdescribed above.

Accordingly, in some embodiments, it is intended not to encompass anypreviously known product, process of making the product, or method ofusing the product such that Applicants reserve the right and herebydisclose a disclaimer of any previously known product, process, ormethod. It is further noted that it is not intended to encompass anyproduct, process, or making of the product or method of using theproduct, which does not meet the written description and enablementrequirements of the USPTO (35 U.S.C. § 112, first paragraph) or the EPO(Article 83 of the EPC), such that Applicants reserve the right andhereby disclose a disclaimer of any previously described product,process of making the product, or method of using the product. It may beadvantageous in the practice various embodiments in compliance with Art.53(c) EPC and Rule 28(b) and (c) EPC. All rights to explicitly disclaimany embodiments that are the subject of any granted patent(s) ofapplicant in the lineage of this application or in any other lineage orin any prior filed application of any third party is explicitlyreserved. Nothing herein is to be construed as a promise.

It is noted that in this disclosure and particularly in the claimsand/or paragraphs, terms such as “comprises”, “comprised”, “comprising”and the like can have the meaning attributed to it in U.S. patent law;e.g., they can mean “includes”, “included”, “including”, and the like;and that terms such as “consisting essentially of” and “consistsessentially of” have the meaning ascribed to them in U.S. patent law,e.g., they allow for elements not explicitly recited, but excludeelements that are found in the prior art or that affect a basic or novelcharacteristic of the invention.

These and other embodiments are disclosed or are obvious from andencompassed by, the following Detailed Description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description, given by way of example, but notintended to limit the invention solely to the specific embodimentsdescribed, may best be understood in conjunction with the accompanyingdrawings.

FIG. 1 —Structural organisation of single costimulatory and fusioncostimulatory domain receptors. A schematic representation of CoStARreceptors set out in the claims is shown. First a CoStAR based on asingle costimulatory receptor, and secondly a fusion CoStAR consistingof a full length costimulatory receptor signalling domain fused to asecond costimulatory domain.

FIG. 2 —Genomic organisation of potential CoStAR configurations—TheCoStAR consists of an antigen binding domain, an optional spacer domainand a costimulatory domain as shown in figure and described in claims.The CoStAR may be expressed: A) alone from a promoter with the CoStARconsisting of a single (Ai) or fusion (Aii) costimulatory receptor; B)may be expressed with an epitope tag (e.g. His tag, DYKDDDDK (SEQ IDNO:449) etc) at the N or C-terminus to enable direct staining of theCoStAR; C) along with a marker gene separated using a 2A cleavagesequence or internal ribosomal entry site (IRES); D) along with a markergene which is expressed from a second promoter; E) along with a proteinof interest such as a chimeric antigen receptor or T-cell receptorseparated using a 2A cleavage sequence or internal ribosomal entry site(IRES); F) along with a protein of interest such as a chimeric antigenreceptor or T-cell receptor which is expressed from a second promoter.It would be clear to an individual with sufficient knowledge that theCoStAR and marker gene/chimeric antigen receptor/T-cell receptor/otherprotein of interest could be expressed in either orientation or 3′(3-prime) or 5′ (5-prime) to one another.

FIG. 3A-3E—Functional activity of CoStAR in T-cells in response toLS174T and LoVo tumour presented antigen. Normal donor T-cellpopulations from donor 1 (3A & 3D), donor 2 (3B) and donor 3 (3C & 3E)were lentivirally engineered to express a CoStAR which targetscarcinoembryonic antigen and magnetically sorted to enrich for thetransgene using CD34 magnetic selection. T-cells were mixed withwild-type un-engineered CEA+ tumour cells (Non-activating tumour) orCEA+ tumour cells engineered to express a cell surface anchored anti-CD3single chain antibody fragment (Activating tumour) at the indicatedeffector to target ratios and IL-2 measured in the supernatant by ELISA.Data obtained using LS174T cells (3A, 3B & 3C) and LoVo (3D & 3E).

FIG. 4A-4D—Effect of CoStAR on T-cell proliferation. 5×10⁵ transducedand non transduced T-cells were mixed with 6.25×10³ or 6.25×10⁴wild-type LoVo or LoVo-OKT3 cells in the presence (4A) or absence (4B)of IL-2 and cell counts made after three days. In another assay underthe same cell ratios T-cells from two donors (4C and 4D) were loadedwith proliferation dye and the number of proliferation cycles the cellshad gone through determined by dye dilution after six days using flowcytometry.

FIG. 5 —IL-2 activity of CoStAR fusion receptors in primary humanT-cells. Normal donor CD8+ T-cells from seven donors (except controlCoStAR is three donors) were lentivirally transduced with the indicatedCEA-targeting CoStARs and IL-2 production assessed after an overnightstimulation in the presence of LoVo-OKT3 cells. The proportion of IL-2positive cells was determined using intracellular flow staining in boththe CD34 negative (CoStAR non-transduced) and CD34+(CoStAR transduced)populations. Asterisks show significant differences between thetransduced and non-transduced populations using paired Wilcoxon signedrank test with * p<0.05

FIG. 6A-6D—Multi parameter analysis of CoStAR activity in primary humanT-cells. Normal donor CD8+ T-cells were lentivirally transduced with theindicated CEA-targeting CoStARs and IL-2 production assessed after anovernight stimulation in the presence of LoVo-OKT3 cells. The proportionof IL-2 (seven donors) (FIG. 6A), IFNγ (seven donors) (FIG. 6B), BCL-xL(five donors) (FIG. 6C) and CD107a (six donors) (FIG. 6D) positive cellswas determined using intracellular flow staining in both the CD34negative (CoStAR non-transduced) and CD34+(CoStAR transduced)populations. Control is a non-specific CA125 targeting CoStAR and isfrom three donors in all instances. Heat maps are averages of all donorswith the intensity of colour related to the percentage of cells positivefor a particular read out under the defined conditions.

FIG. 7 —CD40 enhances IL-2 production from CD28-based CoStARs. Primaryhuman T-cells from three healthy donors were left non-transduced ortransduced with either extracellular domain truncated CD28 (Tr CD28),full length CD28 (FL CD28), or CD28.CD40-based CoStARs harbouring a CEAspecific scFv (MFE23). Transduced cells were selected using a CD34marker gene and expanded prior to analysis. T-cells were mixed at an 8:1effector to target ratio with OKT3 expressing CEA+ LoVo cells for 20hours before analysis of IL-2 production by ELISA.

FIG. 8 —Effect of signalling domain and target antigen onCoStAR-mediated T-cell expansion. T-cells were transduced with eitherDYKDDDDK (SEQ ID NO:449) epitope-tagged CD28 or CD28.CD40 based CoStARsharbouring CA125, FolR or CEA specific scFv, or FolR specific bindingpeptide (C7). T-cells were mixed with OKT3 expressing, CA125+/FolR+/CEA−cell line OVCAR3. The number of transduced cells were counted every 7days up to 21 days, with fresh OVCAR3 cells added following each count.

FIG. 9 —Effect of signalling domain and target antigen on CoStAR−mediated T-cell expansion. T-cells were transduced with either DYKDDDDK(SEQ ID NO:449) epitope-tagged CD28 or CD28.CD40 based CoStARsharbouring CA125, FolR or CEA specific scFv, or FolR specific bindingpeptide (C7). T-cells were mixed with OKT3 expressing CA125+/FolR+/CEA−cell line OVCAR3. The number of transduced cells were counted every 7days up to 21 days, with fresh OVCAR3 cells added following each count.

FIG. 10A-10B—(10A and 10B) CD40 based CoStARs enhance costimulation ofT-cells in a model of TCR-transfer. Primary human T-cells from threehealthy donors were transduced with a CEA specific TCR plus either aDYKDDDK-tagged CD28 or CD28.CD40 based CoStAR harbouring either an MFE(open or closed circles) or CA125 (open squares) specific scFv. T-cellswere mixed at a 1:1 effector:target ratio with CEA+/CA125− H508 cellsand intracellular cytokine staining performed to determine the number ofresponding CD4+ or CD8+ T-cells in the TCR+/CoStAR+, TCR+/CoStAR−,TCR−/CoStAR+ and TCR−/CoStAR− populations. A 2-way ANOVA (Tukeys test)was performed to determine significant differences in activity: *p>0.05,** p>0.01, *** p>0.001, **** p>0.0001.

FIG. 11 depicts enrichment and expansion of primary human T-cellstransduced to express costimulatory molecules. MFE23 is a single chainFv antibody that has a high affinity for carcinoembryonic antigen (CEA).Primary human T-cells were mock transduced or transduced with MFE23.CD28or MFE23.CD28.CD40 CoStAR, each harboring a CD34 marker gene separatedby a 2A cleavage peptide. Following in vitro culture cells were enrichedfor CD34 using MACS™ paramagnetic selection reagents (Miltenyi Biotech)and then the cells expanded in number using irradiated feeder cells.Exemplary plots from one of three donors are shown.

FIG. 12A-12D depicts expansion of T-cells transduced with costimulatorymolecules in response to stimulation and exogenous IL-2. Cells were mocktransduced or transduced with MFE23.CD28 or MFE23.CD28.CD40 CoStAR andcocultured with LoVo-OKT3 cells at an 8:1 effector:target ratio in thepresence (200 IU/ml) or absence of exogenous IL-2. At days 1, 4, 7, 11and 18 cells were taken and the number of viable T-cells enumerated byusing anti-CD2 reagents on a MACSQuant flow cytometer. (12A) In theabsence of stimulation by tumor and IL-2 cells declined in number aswould be expected. (12B) In the absence of stimulation but presence ofIL-2 there was a more apparent survival of the cells, but no specificgrowth. (12C) In the presence of tumor, but absence of IL-2 mock cellsdid not show specific survival. MFE23.CD28 CoStAR mediated an apparentdoubling in expansion over the first four days followed by decline.MFE23.CD28.CD40 mediated a greater expansion up to day 7 followed by asteady decline. (12D) Under the same conditions but in the presence ofIL-2 both mock and MFE23.CD28 transduced cells demonstrated a 20-foldexpansion over 18 days, whereas MFE23.CD28.CD40 cells expanded by over60-fold. Thus CD28.CD40 based receptors demonstrate superior expansionand survival under conditions of stimulation both in the presence andabsence of exogenous IL-2.

FIG. 13A-13M depicts cytokine production by mock, MFE23.CD28 orMFE23.CD28.CD40 engineered T-cells. Bead array analysis was performed onsupernatants obtained from T-cell/tumour cocultures. Engineered T-cellswere incubated at a 1:1 effector:target ratio with LoVo-OKT3 cells for24 hours and supernatant collected. Conditioned supernatant was alsocollected from an equal number of T-cells alone, or LoVo-OKT3 cellsalone. Cytokine production was analysed using a Legendplex™ HumanTH1/TH2 cytokine panel (Biolegend). (13A) IL-2; (13B) IFN-γ; (13C) TNFα;(13D) IL-4; (13E) IL-5; (13F) IL-13; (13G) IL-17A; (13H) IL-17F; (13I)IL-22; (13J) IL-6; (13K) IL-10; (13L) IL-9; (13M) IL-21. Cytokines wereeither very low or undetectable in media from T-cells or tumour alone.When cocultured with tumor, cytokine production was enhanced. MFE23.CD28enhanced production of IL-2, IL-5, IL-17A/17F, IL-10, IL-9 and IL-21compared to mock. MFE23.CD28.CD40 also enhanced production of TNFα,IL-13 and IL-22. MFE23.CD28.CD40 and further enhanced the production ofa number of cytokines greater than that provided by MFE23.CD28 (IL-2,IL-9 and IL-17F), as well as reducing the production of some cytokinesbelow the levels seen with MFE23.CD28 (IL-5 and IL-10). Together thisdata demonstrates that addition of CD40 to CD28-based costimulatoryreceptors enhances and/or modulates their specific activity with respectto chemokine production.

FIG. 14A-14M depicts an analysis of chemokines using a Legendplex™ HumanPro inflammatory chemokine panel. (14A) IL-8 (CXCL8); (14B) IP-10(CSCL10); (14C) Eotaxin (CCL11); (14D) TARC (CCL17); (14E) MCP-1 (CCL2);(14F) RANTES (CCL5); (14G) MIP-1a (CCL3) (14H) MIG (CXCL9) (14I) ENA-78(CXCL5) (14J) MIP-3α (CCL20) (14K) GROα (CXCL1) (14L) I-TAC (CXCL11)(14M) MEP-10 (CCL4). Chemokines were either very low or undetectable inmedia from T-cells alone. When cocultured with tumor, chemokineproduction was enhanced. MFE23.CD28 enhanced production of CXCL5,CXCL10, CXCL11, CCL17 and CCL20 compared to mock. MFE23.CD28.CD40 alsoenhanced production of CCL2, CXCL1 and CXCL9. MFE23.CD28.CD40 furtherenhanced the production of a number of cytokines greater than thatprovided by MFE23.CD28 (CXCL1, CXCL9, CXCL10, CXCL11, CCL17, CCL2,CXCL9, CCL5 and CCL20), as well as reducing the production of somecytokines below the levels seen with MFE23.CD28 (CCL4). Together thisdata demonstrates that addition of CD40 to CD28-based costimulatoryreceptors enhances and/or modulates their specific activity with respectto chemokine production.

FIG. 15A-15H depicts functional activity of ovarian CoStAR engineeredcells using a CoStAR harbouring a FolR or CA125 reactive scFv (MOV19 &196-14 respectively). Human folate receptor alpha (FolR) represents asuitable target for a number of tumours including ovarian, head andneck, renal and lung and CA125 represents an alternative target forovarian cancer. Primary human T-cells from six healthy donors wereengineered with either 196-14.CD28, 196-14.CD28.CD40, MOV19.CD28 orMOV19.CD28.CD40 receptors, all harbouring a DYKDDDDK epitope tag fordetection. Transduced cells were mixed with FolR+/CA125+ OVCAR-OKT3cells before analysis of effector activity using intracellular stainingin the epitope tag positive and negative populations. Specificenhancement of effector activity determined by production of IL-2 (15Aand 15B), TNFα (15C and 15D), CD137 (15E and 15F), and BCL-xL (15G and15H) was observed in in CD28 and CD28.CD40 engineered cells in responseto both CA125 and FolR, except for specific BCL-xL induction byMOV19.CD28 which was not observed compared to MOV19.CD28.CD40.

FIG. 16A-16F depicts three TIL populations mock transduced or engineeredwith MOV19.CD28.CD40 CoStAR and then mixed with patient matched tumordigest. The donor tumors displayed varying levels of FolR on the digest,ranging from negative (16A), low expression (16B) to high expression(16C). Mock and CoStAR negative TIL in the CoStAR engineered populationsof TIL matched for the FolR negative digest demonstrated similar levelsof CD137 upregulation following tumor coculture which was not enhancedby the presence of CoStAR (16D). In the TIL exposed to FolR lowexpressing digest there was an enhancement in activity in the CoStAR+cells compared to CoStAR−, with CD137 expression increasing from <10%to >20% (16E). In the TIL exposed to FolR high tumor digest there was anincrease in activity from around 20% in the CoStAR− population, up toapproximately 50% in the CoStAR+ population (F).

FIG. 17A-17C depicts enhancement of effector functions. A FolR targetingCoStAR enhanced CD137 expression from ˜20% to ˜50% (17A), TNFαproduction from 10% to 15% (17B) and IL-2 production from 2% to 5%.(17C) in response to FolR+ tumor digest.

FIG. 18A-18F depicts soluble ligand does not inhibit effector functions.T-cells from three healthy donors were engineered with MOV19.CD28 orMOV19.CD28.CD40 CoStAR and activated with either immobilised OKT3,providing stimulation in the absence of FolR, or with OvCAR-OKT3, toprovide TCR and CoStAR activity. BCL-xL activity was increased frombetween 10 and 20% across the three donors following OKT3 stimulation(18A) whereas IL-2 was increased between 0 and 12% (18B) and TNFαincreased between 0 and 20% (18C). The presence of exogenous solubleFolR did not enhance any of these particular effector functions. In thepresence of OvCAR-OKT3 BCL-XL induction was enhanced by ˜20% in CD28CoStAR but by ˜35% in CD28.CD40 CoStAR (18D), IL-2 induction wasenhanced by ˜20% in CD28 CoStAR but 30-50% in CD28.CD40 CoStAR (18E) andTNFα production was enhanced by 20-30% in CD28 CoStAR and 25-50% inCD28.CD40 CoStAR (18F). Exogenous soluble FolR did not have aninhibitory effect on any of these effector functions.

FIG. 19 depicts surface expression of anti-MSLN CoStAR expression on thesurface of HD T cells. Transduced and non-transduced (MOCK) cellsunderwent a rapid expansion protocol (REP) and were assessed fortransduction efficiency either via surface detection of the marker genetCD34 (truncated CD34) or CoStAR molecule using an anti-CD34-APC (black)or anti-MSLN-PE (red) antibody, respectively. The results represent 3biological replicates.

FIG. 20A-20C depicts cytokine expression in healthy donor (HD) T cellstransduced with anti-MSLN CoStARs and cocultured with OVCAR-3 celllines. CoStARs comprising combinations of six different anti-MSLN scFvs(SS1, M5, HN1, M912, huYP218, P4) and three differentspacer/transmembrane domains (CD28, N-terminal truncated CD28, CD8) werecompared. Structural details are provided in Table 8, Table 9, and Table10. Cytokine concentrations for (20A) IL-2 (20B) IFN7 and (20C) TNFαfollowing cocultures with OVCAR-3 or OVCAR3-OKT3 cell lines are shown.Non-treated T cells were used as a control. The results represent 1-3biological replicates with 3 technical replicates each.

FIG. 21A-21C depicts cytokine expression in healthy donor (HD) T cellstransduced with anti-MSLN CoStARs and cocultured with K562 cell lines.CoStARs comprising combinations of six different anti-MSLN scFvs (SS1,M5, HN1, M912, huYP218, P4) and three different spacer/transmembranedomains (CD28, N-terminal truncated CD28, CD8) were compared. Structuraldetails are provided in Table 8, Table 9, and Table 10. Cytokineconcentrations for (21A) IL-2 (21B) IFN7 and (21C) TNFα followingcocultures with K562-MSNL or K562-MSNL-OKT3 cell lines are shown.Non-treated T cells were used as a control. The results represent 1-3biological replicates with 3 technical replicates each.

FIG. 22 depicts surface expression of MFE23 scFV anti-CEA CoStARsexpressed with six different secretion signal peptides (OSM1, CD8, CD2,IL2, GMCSF, hIgGκ). Structural details are provided in Table 11.Following expansion, cells were assessed for transduction efficiencyeither via surface detection of the marker gene truncated CD34 (tCD34)or CoStAR molecule using an anti-CD34-APC (black bars) or using aprimary rhCEACAM5-Fc antibody with a secondary anti-IgG-Fc-PE (greybars) antibody, respectively.

FIG. 23 depicts surface expression of CoStARs comprising six differentanti-CEA scFvs (MFE23, MFE23(Q>K), hMFE23, CEA6, BW431/26, hT84.66).Structural details are provided in Table 12. Following expansion, cellswere assessed for transduction efficiency either via surface detectionof the marker gene truncated CD34 (tCD34) or CoStAR molecule using ananti-CD34-APC (black bars) or using a primary rhCEACAM5-Fc antibody witha secondary anti-IgG-Fc-PE (grey bars) antibody, respectively.

FIG. 24A-24C depicts cytokine production by healthy donor (HD) T cellstransduced with anti-CEA CoStARs (MFE23, MFE23(Q>K), hMFE23, CEA6,BW431/26, hT84.66) and cocultured with LoVo cell lines. Structuraldetails are provided in Table 12. Cytokine concentrations are shown for(24A) IL-2 (24B) IFN7 and (24C) TNFα following cocultures with Lovo orLovo-OKT3 cell lines. Non-treated T cells were used as a negativecontrol.

FIG. 25A-25C depicts cytokine production in healthy donor (HD) T cellstransduced with anti-CEA CoStARs (MFE23, MFE23(Q>K), hMFE23, CEA6,BW431/26, hT84.66) and cocultured with K562 cell lines. Cytokineconcentrations are shown for (25A) IL-2 (25B) IFN7 and (25C) TNFαfollowing cocultures with K562.CEACAM5 (signal 2) or K562.CEACAM5.OKT3(signal 1+2) cell lines. Non-treated T cells were used as a negativecontrol.

FIG. 26 depicts surface expression of hMFE23 scFV anti-CEA CoStARsexpressed with three different spacer/transmembrane domains. Structuraldetails are provided in Table 13. Following expansion, cells wereassessed for transduction efficiency via surface detection of the markergene truncated CD34 (tCD34) using an anti-CD34-APC (black bars) or theCoStAR molecule using a primary rhCEACAM5-Fc antibody with a secondaryanti-IgG-Fc-PE antibody (grey bars).

FIG. 27A-27C depicts cytokine production by the MFE23 scFV anti-CEAspacer variants. Cytokine concentrations for (27A) IL-2 (27B) IFN7 and(27C) TNFα following cocultures with Lovo or Lovo.OKT3 cell lines areshown. Non-treated T cells were used as a control.

FIG. 28 depicts anti-CEA CoStARs comprising an hMFE23 CEA-binding domainwith intracellular signalling domains comprising CD40, CD134, CD137,CD2, ICOS, DAP10 and NTRK1 signaling elements.

FIG. 29 depicts surface expression of hMFE23 scFV anti-CEA CoStARscomprising domain combinations depicted in FIG. 28 and detail in Table14. Following expansion, cells were assessed for transduction efficiencyvia surface detection of the marker gene truncated CD34 (tCD34) using ananti-CD34-APC (black circles) or the CoStAR molecule using a primaryrhCEACAM5-Fc antibody with a secondary anti-IgG-Fc-PE antibody (redcircles). The results represent three biological replicates.

FIG. 30 depicts T cell phenotypes of CoStAR transfected HD T cells inthree separate donors. Cells were sorted using CD34 microbeads andunderwent a rapid expansion protocol (REP) for 14 days. Followingoutgrowth and REP, 1×10⁵ cells were assessed for the differentiationsubtype using flow cytometry. Tcm, central memory T cell; Tem, effectormemory T cell; Tn, naïve T cell; Tscm; stem cell memory T cell; Tte,terminal effector T cell. See Table 15 for T cell subtype definitions.

FIG. 31A-31C depicts cytokine production by hMFE23 scFV anti-CEA CoStARtransduced HD T cells cocultured with K562 cell lines. Cytokineconcentrations for (31A) IL-2, (31B) IFNγ, and (31C) TNFα are shownfollowing cocultures with K562.CEACAM5 (signal 2) or K562.CEACAM5.OKT3(signal 1+2) cell lines. Non-treated T cells were used as a control.

FIG. 32A-32C depicts cytokine expression in HD T cells transduced withhMFE23 scFV anti-MSLN CoStARs and cocultured with K562 cell lines.Frequency of (32A) IL-2, (32B) IFNγ, and (32C) TNFα expressing cells isshown following cocultures with K562.CEACAM5 (signal 2) orK562.CEACAM5.OKT3 (signal 1+2) cell lines. Non-treated T cells were usedas a control.

FIG. 33 depicts proliferation of HD T cells from transduced with hMFE23scFV anti-MSLN CoStARs cocultured with K562.CEACAM5.OKT3 (signal 1+2)cell lines. HD T cells were procured from two donors. The figuresrepresent fold expansion of input cells.

FIG. 34 depicts proliferation of HD T cells from transduced with hMFE23scFV anti-MSLN CoStARs cocultured with K562.CEACAM5.OKT3 (signal 1+2)cell lines. HD T cells were procured from two donors. The figuresrepresent fold expansion of input cells on Day 6 post stimulation.

FIG. 35A-35B depicts TRAF and TRAF-like binding sites and motifs andsignalling pathways. 35A. CoStAR CD40 intracellular domain showing TRAFbinding sites and signalling. 35B. CoStAR comprising an IProx domain.

FIG. 36A-36B depicts the effect of mutations in CoStAR CD40intracellular signaling domain on cytokine secretion and long termsurvival and proliferation of CD28.CD40 CoStAR transduced T cellscocultured with LoVo.OKT3. Cells of three donors were activated withDynabeads and transduced with WT CD28.CD40 (CTP194), CD28.CD40containing TRAF2 binding site mutation SVQE>AVQA (CTP195), TRAF2/TRAF3binding site mutation PVQET>AVAEA (CTP196), TRAF6 binding site mutationPQEINF>AQAINF (CTP197), Q263A (CTP199), or mock transduced. (36A) IL-2was measured in supernatants collected 24 hours after coculture inabsence of IL-2 with LoVo or LoVo.OKT3.GFP tumor cells. (36B) Viabilityand absolute count were assessed after 6-8 days and live T cells wererechallenged for an additional week with fresh LoVo.OKT3.GFP tumorcells. At the end of the long-term coculture, the viability and absolutecount were measured, and the fold expansion was calculated. Data shownas mean+/−SEM of n≤3 donors analysed in triplicates.

FIG. 37A-37B depicts percentage of TIL (37A) and total TIL counts (37B)based on CD2+ stain in thawed OC samples at day 1.

FIG. 38 depicts growth of non-Td and Td TILs. Cells were counted usingCD2 and DRAQ7 staining and acquisition on the Novocyte 3005. Cell countsat the end of REP on day 25 are graphically represented. Shapescorresponding to the each of the 5 ovarian cancer samples are depicted.Filled shapes are Non-Td and open shapes are Td TILs. Statisticalanalysis was performed using a paired t-test.

FIG. 39A-39D depicts transduction efficiency and viral integrations percell of CoStAR modified TILs. Nontransduced (Non-Td) and anti-FOLR1CoStAR transduced (Td) TILs were assessed for the transductionefficiency on day 25 post REP in the CD3+ (39A), CD4+ (39B) and CD8+(39C) cell populations. The viral copy number (VCN) was assessed todetermine viral integrations (39D). Shapes corresponding to the each ofthe 5 ovarian cancer samples are depicted. Filled shapes are non-Td andclear shapes are Td TILs.

FIG. 40 depicts CD4 and CD8 populations in post-REP TILs. Nontransduced(Non-Td) and anti-FOLR1 CoStAR− transduced (Td) and anti-FOLR1 CoStAR+Td TILs were assessed for the CD4 and CD8 composition on D25 post-REPusing flow cytometry. Statistical analysis was performed using a two-wayANOVA with matched Tukey's multiple comparisons post-test.

FIG. 41A-41C depicts the effect of CoStAR modification on thedifferentiation status of TILs. Nontransduced (Non-Td), anti-FOLR1CoStAR− transduced (Td) TILs and anti-FOLR1 CoStAR+ Td TILs wereassessed for their differentiation status on D25 post-REP from total Tcell (41A), CD4+ (41B) and CD8+ (41C) cell populations. Statisticalanalysis was performed using a two-way ANOVA with matched Tukey'smultiple comparisons test. Statistical significance was observed for theTscm population between the anti-FORL1 CoStAR− Td TILs and anti-FOLR1CoStAR+ Td TILs for CD3+ (A) and CD4+ (B) populations. *p<0.05

FIG. 42A-42B depicts effects of CoStAR modification of TILs onco-inhibitory or co-stimulatory marker expression. Nontransduced(Non-Td), anti-FOLR1 CoStAR-transduced (Td) TILs and anti-FOLR1 CoStAR+Td TILs were assessed for the expression of co-inhibitory andco-stumulatory markers in CD4+ (42A) and CD8+ (42B) cell populations.Shapes corresponding to the each of the 5 ovarian cancer samples aredepicted regardless of the filling. Black, dark grey and light grey barsrepresent Non-Td TILs, anti-FOLR1 CoStAR− Td and anti-FOLR1 CoStAR+ TdTILs, respectively. Statistical analysis was performed using a Two-wayANOVA with a matched Tukey's multiple comparisons post-test. *p<0.05

FIG. 43A-43B depicts effects of CoStAR modification on TCRαβ, TCRγδ andTreg frequency in TILs. Nontransduced (Non-Td), anti-FOLR1 CoStAR−transduced (Td) TILs and anti-FOLR1 CoStAR+ Td TILs were assessed forthe frequency of TCRαβ (CD3+TCRαβ+), and TCRγδ (CD3+TCRγδ+) in CD3+ cellpopulations (43A). The frequency of Tregs in the CD4+ subpopulation wasalso assessed (43B). Shapes corresponding to the each of the 5 ovariancancer samples are depicted regardless of the filling. Black, dark greyand light grey bars represent Non-Td TILs, anti-FOLR1 CoStAR− Td TILsand anti-FOLR1 CoStAR+ Td TILs, respectively. Statistical analysis wasperformed using a two-way ANOVA with matched Tukey's multiplecomparisons post-test (43A) and a one-way ANOVA with a Friedman'spost-test (43B).

FIG. 44A-44C depicts effects of CoStAR modification of TIL on cytokineproduction upon mitogenic activation. Nontransduced (Non-Td), anti-FOLR1CoStAR-transduced (Td) TILs and anti-FOLR1 CoStAR+ Td TILs were assessedfor the production of cytokines upon 4 hour stimulation with PMA (50ng/mL)/Ionomycin (1 μg/mL) in CD3+ (44A), CD4+ (44B) and CD8+ (44C) cellpopulations. Shapes corresponding to the each of the 5 OC samples aredepicted regardless of the filling. Black, dark grey and light grey barsrepresent Non-Td TILs, anti-FOLR1 CoStAR− Td TILs and anti-FOLR1 CoStAR+Td TILs, respectively. Statistical analysis was performed using atwo-way ANOVA with matched Tukey's multiple comparisons post-test. *p≤0.05, *** p≤0.001.

FIG. 45 depicts Expression of FOLR1 on OC digest cells. Cryopreservedautologous tumor digest was thawed and analyzed by flow cytometry todetermine the surface expression of FOLR1 of each donor. All 5 donorsused in the study are shown. Abbreviations: OC, ovarian cancer, FOLR1,folate receptor alpha; FOLR1 PE, anti-FOLR1 antibody conjugated to PE;PE, phycoerythrin.

FIG. 46A-46D depicts effect of CoStAR modification on cytokine producingcells upon coculture with autologous tumors. Non-Td and anti-FOLR1CoStAR Td TILs were cocultured with autologous tumor digests for 16hours and intracellular flow cytometry was performed to evaluate theproportion of cells producing cytokines. The frequency of IL-2 positive(46A) CD4 and (46B) CD8 TILs as well as the frequency of TNFα positive(46C) CD4 and (46D) CD8 TILs were assessed. Results represent 5biological replicates with 3 technical replicates each. Statisticalanalysis was performed using a two-way ANOVA with Tukey's multiplecomparisons test. *P<0.05.

FIG. 47A-47E depicts the effect of CoStAR modification on cytokinesecretion upon coculture with autologous tumor digests expressing FOLR1.Non-Td and anti-FOLR1 CoStAR transduced (Td) TILs were cocultured withautologous tumor digest for 24 hours, following which supernatants werecollected and analyzed for cytokine secretion using an MSD immunoassay.The graphs represent measured concentrations of (47A) IL-2, (47B) TNFα,(47C) IL-13 and (47D) IFN7 in coculture supernatants. (47E) Correlationbetween IFN7 concentration and FOLR1 expression by autologous digest asdetermined in FIG. 1 . Results represent 5 biological replicates with 3technical replicates each. Statistical analysis was performed using atwo-way ANOVA with Sidak's multiple comparisons test. Correlationanalysis was performed using a simple linear regression. *P<0.05,**P<0.01, ***P<0.001.

FIG. 48 depicts assessment of MHC-dependent CoStAR functionality incocultures with autologous tumor digests. Non-Td and anti-FOLR1 CoStARtransduced (Td) TILs were cocultured with autologous tumor digests inthe presence of MHC blocking reagents or isotype controls for 24 hours.The supernatants were then analyzed for IFN7 cytokine production usingthe MSD immunoassay. Results represent 3 biological replicates with 3technical replicates each. Statistical analysis was performed using atwo-way ANOVA with Tukey's multiple comparisons test.

FIG. 49A-49B depicts the effect of CoStAR modification in cytokineproducing cell frequencies upon coculture with engineered cell lines.Non-Td and anti-FORL1 CoStAR transduced (Td) TILs were cocultured withengineered target cell lines for 16 hours and cytokine producing cellswere measured using intracellular flow cytometry. Frequency of IL-2positive (49A) in CD4+ (top) and CD8+ (bottom) TILs following coculturewith K-562 (left) and OVCAR-3 (right) derived lines. Frequency of TNFαpositive (49B) in CD4+ (top) and CD8+ (bottom) TILs following coculturewith K-562 (left) and OVCAR-3 (right) derived lines. Results represent 5biological replicates with 3 technical replicates each. Statisticalanalysis was performed using a two-way ANOVA with Tukey's multiplecomparisons test. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

FIG. 50A-50D depicts the effect of CoStAR modification in cytokinesecretion upon coculture with engineered cell lines. Non-Td andanti-FOLR1 CoStAR transduced (Td) TILs were cocultured with engineeredtarget cell lines for 24 hours and MSD immunoassay was performed toevaluate the concentration of cytokines secreted. Cytokineconcentrations for (50A) IL-2, (50B) TNFα, (50C) IL-13 and (50D) IFN7following cocultures with (left) K-562, and (right) BA/F3 derived celllines are shown. The results represent 5 biological replicates with 3technical replicates each. Statistical analysis was performed using atwo-way ANOVA with Tukey's multiple comparisons test. *P<0.05, **P<0.01,***P<0.001, ****P<0.0001.

FIG. 51A-51C depicts assessment of the impact of CoStAR modification onthe cytotoxic capacity of TILs against OKT3 bearing targets. Non-Td andanti-FOLR1 CoStAR transduced (Td) TILs were cocultured with BA/F3 orOVCAR-3 engineered cell lines and the cytotoxicity was assessed using aflow cytometry based (51A) and an xCELLigence-based assay (51B and 51C),respectively. (51B) E:T ratio of 1:5 and (51C) E:T ratio of 1:30.Results represent 5 biological replicates with 3 technical replicateseach. Statistical analysis was performed using a two-way ANOVA withTukey's multiple comparisons test for flow cytometry data and 1-wayANOVA with Tukey's multiple comparisons test for xCELLigence data.*P<0.05, **P<0.01.

FIG. 52A-52D depicts expansion of NSCLC TILs with >80% viability and˜100-200 million cells, 40-55% CoStar transduction in CD3+ T cellsand >90% CD3+ T cell purity.

FIG. 53 depicts expansion of NSCLC TILs with >80% viability. There wasno difference in CoStAR transduced and nontransduced populations interms of viability and cell expansion.

FIG. 54 depicts expansion of NSCLC TILs with 50-500 million cells atharvest. There was no difference in CoStAR transduced and nontransducedpopulations in terms of viability and cell expansion.

FIG. 55 depicts T cell populations of seven patient samples.

FIG. 56 depicts CD3 vs CD56 which marks CD3+ as T cells and CD3-CD56+ asNK cells.

FIG. 57 depicts quantification of non-transduced and CoStAR transduced Tcells.

FIG. 58 depicts CoStAR staining on T cell subsets.

FIG. 59A-59D depicts significant increase in viability of ovarian cancerTILs during REP and >90% viability at the end of the manufacturingprocess, with 100-300 million cells at harvest.

FIG. 60A-60D depicts renal cancer TILs demonstrated consistently >50%viability during outgrowth and >90% viability at the end of themanufacturing process, with 100-350 million cells at harvest.

FIG. 61A-61B depicts IHC performed with anti mesothelin antibody (clone5B2) with a Dako Autostainer Link 48 according to standard protocols.Scoring was evaluated by a pathologists interpretation of % cellspositive for 5B2 binding. H-scores were calculating the % cells positivefor 5B2 binding multiplied by a pathologists estimation of: 0 (nostaining) 1 (weak staining), 2 (moderate staining) or 3 (strongstaining).

FIG. 62 depicts surface expression of scFV anti-MSNL CoStARs comprisingof different scFV domains but retaining all other domains acrossconstructs. Cells were assessed for transduction efficiency on day fourpost transduction, via surface detection of the marker gene truncatedCD34 (tCD34) using an anti-CD34-APC (black bars) or the CoStAR moleculeusing a primary MSNL-PE antibody (grey bars). The results represent onebiological replicate.

FIG. 63A-63B depicts dot plots of the surface expression of scFVanti-MSNL CoStARs comprising of different scFV domains but retaining allother domains across constructs. Cells were assessed for transductionefficiency on day four post transduction, via surface detection of themarker gene truncated CD34 (tCD34) using an anti-CD34-APC (y axis) orthe CoStAR molecule using a primary MSNL-PE antibody (x axis).

FIG. 64 depicts surface expression of scFV anti-MSNL CoStARs comprisingof different scFV domains but retaining all other domains acrossconstructs. Cells were assessed for transduction efficiency on day fourpost transduction via surface detection of the marker gene truncatedCD34 (tCD34) using an anti-CD34-APC (black bars, black circles) or theCoStAR molecule using a primary MSNL-PE antibody (grey bars, redcircles). Each graph represents the transduction efficiency on CD3, CD4and CD8 populations. The results represent one biological replicate.

FIG. 65 depicts surface expression of scFV anti-MSNL CoStARs comprisingof different scFV domains but retaining all other domains acrossconstructs. Cells were sorted using CD34 microbeads and placed into arapid expansion protocol (REP) for 14 days. Following expansion,anti-CD34-APC (black bars, black circles) or the CoStAR molecule using aprimary MSNL-PE antibody (grey bars, red circles). Each graph representsthe transduction efficiency on CD3, CD4 and CD8 populations. The resultsrepresent one biological replicate.

FIG. 66 depicts surface expression of scFV anti-MSNL CoStARs comprisingof different scFV domains but retaining all other domains acrossconstructs. Cells were sorted using CD34 microbeads and placed into arapid expansion protocol (REP) for 14 days. Following expansion,anti-CD34-APC (black circles) or the CoStAR molecule using a primaryMSNL-PE antibody (red circles). Each graph represents the transductionefficiency on CD3 cell population. The results represent 3 biologicalreplicates.

FIG. 67 depicts an experimental plan where donor PBMCs were transducedwith a lentivirus for 1 of the 6 MSLN targeting CoStARs. Transducedcells were allowed outgrowth, followed by CD34 selection and a 12 dayREP, followed by co-culture with the naturally MSLN and OKT3 expressingOvcar3 cell line, where cytokine release by CoStAR expressing cells wasevaluated

FIG. 68 depicts the gating strategy and MSNL detection on OVCAR-3 andOVCAR-3-OKT3 detection using flow cytometry. The cells were stainedusing an anti-MSNL APC antibody

FIG. 69A-69B depicts cytokine expression in HD T cells transduced withscFV anti-MSLN CoStARs and cocultured with K562 cell lines. The assessedconstructs varied in the scFV and the spacer domains as shown below.Frequency of IL-2, IFNγ, and TNFα expressing cells is shown followingcocultures with OVCAR-3 (signal 2) or OVCAR-3-OKT3 (signal 1+2) celllines. Non-treated T cells were used as a control. The results represent3 biological replicates.

FIG. 70A-70B depicts cytokine expression in HD T cells transduced withscFV anti-MSLN CoStARs and cocultured with K562 cell lines. The assessedconstructs varied in the scFV and the spacer domains as shown above.Frequency of (A) IL-2, (B) IFNγ, and (C) TNFα expressing cells is shownfollowing cocultures with K562.MSNL (signal 2) or K562.MSNL.OKT3 (signal1+2) cell lines. Non-treated T cells were used as a control. The resultsrepresent 3 biological replicates.

FIG. 71 depicts fold expansion of unmodified and anti-CEA CoStARmodified TILs at the end of manufacture for two CoStAR constructs.

FIG. 72 depicts transduction efficiency of TILs with two anti-CEA CoStARconstructs at the end of manufacture (Day 21).

FIG. 73 depicts transduction efficiency of TILs generated from CRC,NSCLC, ovarian tumors, and melanoma with two anti-CEA CoStAR constructsat the end of manufacture (Day 21).

FIG. 74 depicts memory phenotype of TILs generated from CRC, NSCLC,ovarian tumors, and melanoma and transduced with two anti-CEA CoStARconstructs at the end of manufacture (Day 21).

FIG. 75 depicts cytokine expression in TILs derived from CRC tumorstransduced with two anti-CEA CoStAR constructs and incubated with targetcells K562, K652-OKT3, K562-CEACAM5, K562-OKT3-CEACAM5, or T cellsalone.

FIG. 76 depicts cytokine expression in TILs derived from NSCLC tumorstransduced with two anti-CEA CoStAR constructs and incubated with targetcells K562, K652-OKT3, K562-CEACAM5, K562-OKT3-CEACAM5, or T cellsalone.

FIG. 77 depicts cytokine expression in TILs derived from OV-9662 cellsand melanoma transduced with two anti-CEA CoStAR constructs andincubated with target cells K562, K652-OKT3, K562-CEACAM5,K562-OKT3-CEACAM5, or T cells alone.

FIG. 78 depicts cytokine expression in TILs derived from melanoma cellstransduced with two anti-CEA CoStAR constructs and incubated with targetcells K562, K652-OKT3, K562-CEACAM5, K562-OKT3-CEACAM5, or T cellsalone.

FIG. 79 depicts fold expansion and transduction efficiency in CRC11959cells transduced with 1 of 2 anti-CEA CoStAR constructs and subsequentlyincubated with K562-OKT3-CEACAM5 cells. Fold expansion was evaluatedfrom 1-4 or 1-6 weeks. Transduction efficiency was evaluated from 0-21or 0-28 days.

FIG. 80 depicts fold expansion and transduction efficiency in NSCLC 9332cells transduced with 1 of 2 anti-CEA CoStAR constructs and subsequentlyincubated with K562-OKT3-CEACAM5 cells. Fold expansion was evaluatedfrom 1-6 weeks. Transduction efficiency was evaluated from 0-28 days.

FIG. 81 depicts fold expansion and transduction efficiency in OV 9662cells transduced with 1 of 2 anti-CEA CoStAR constructs and subsequentlyincubated with K562-OKT3-CEACAM5 cells. Fold expansion was evaluatedfrom 1-4 weeks. Transduction efficiency was evaluated from 0-21 days.

FIG. 82 depicts fold expansion and transduction efficiency in Mel CC50,Mel 11909, and Mel 17614 cells transduced with 1 of 2 anti-CEA CoStARconstructs and subsequently incubated with K562-OKT3-CEACAM5 cells. Foldexpansion was evaluated from 1-4 weeks. Transduction efficiency wasevaluated from 0-21 days.

FIG. 83 depicts the amino acid sequences comprising the CEA.ICOS.CD40CoStAR.

FIG. 84A-84C depicts the amino acid sequences comprising the MSLNtargeting CoStARs CTP224-CTP229 as well as the MSLN targeting scFvsequences for SS1, M5, HN1, M912, huYP218, and P4.

DETAILED DESCRIPTION

Costimulatory receptors comprising a CD40 signaling domain display noveland improved activity profiles. The activity profiles can be modulatedby selecting an intracellular domain of a receptor protein for joiningto the CD40 signaling domain and/or by selecting elements of the CD40signaling domains to join to the intracellular domain of a receptorprotein. Provided herein are recombinant costimulatory antigen receptors(CoStARs) comprising: (i) a disease- or tumor-associated antigen bindingdomain, (ii) a first intracellular segment comprising an intracellularsignaling domain of a receptor protein, and (iii) a second intracellularsignaling domain of a CD40 receptor protein or signal transducingfragment thereof. Optionally, the CoStAR comprises an extracellularsegment of a stimulatory receptor protein. In some embodiments, theextracellular segment of the stimulatory receptor protein is capable ofbinding ligand. In some embodiments, the extracellular segment of astimulatory receptor protein is truncated and does not bind ligand. Insome embodiments the extracellular segment of the stimulatory receptorprotein operates as an adjustable length spacer allowing the disease- ortumor-associated antigen binding domain to be located away from thesurface of the cell in which it is expressed for example to form a moreoptimal immune synapse. In some embodiments, the extracellular segmentof a stimulatory receptor protein and the first intracellular segmentcomprise segments of the same receptor protein. In some embodiments, theextracellular segment and the first intracellular segment comprisesegments of different receptor proteins. The CoStARs comprise anintervening transmembrane domain between the disease or tumor antigenbinding domain and the first intracellular domain. When an extracellularsegment of a stimulatory receptor protein is present, the transmembranedomain is intervening between the extracellular segment and the firstintracellular signaling domain.

In some embodiments, the MSLN targeting CoStAR comprises anh-oncostatin-M signal peptide, a scFv, a linker, a spacer, atransmembrane domain, and an CD28.CD40 intracellular signaling domain.In some embodiments, the fusion protein comprises one or more of: anh-oncostatin-M signal peptide, a scFv, a linker, a spacer, atransmembrane domain, and an CD28.CD40 intracellular signaling domain.

In some embodiments the MSLN targeting CoStAR and/or fusion proteincomprises SEQ ID NO: 192, 210, 228, 246, 264, or 282. In someembodiments the MSLN targeting CoStAR and/or fusion protein comprisesthe first sequence of SEQ ID NO: 1, connected to the second sequence ofany one of SEQ ID NO: 185-191 connected to the third sequence of SEQ IDNO: 18, connected to the fourth sequence of SEQ ID NO: 19, connected tothe fifth sequence of SEQ ID NO: 25, connected to the sixth sequence ofSEQ ID NO: 32. See FIG. 84 for sequences.

In some embodiments, the CEA targeting CoStAR comprises anh-oncostatin-M signal peptide, a scFv, a linker, an ICOS domain, and anCD40 intracellular signaling domain. In some embodiments, the fusionprotein comprises one or more of: an h-oncostatin-M signal peptide, ascFv, a linker, an ICOS domain, and an CD40 intracellular signalingdomain.

In some embodiments the CEA targeting CoStAR and/or fusion proteincomprises SEQ ID NO: 348. In some embodiments the CEA targeting CoStARand/or fusion protein comprises the first sequence of SEQ ID NO: 1,connected to the second sequence SEQ ID NO: 12 connected to the thirdsequence of SEQ ID NO: 18, connected to the fourth sequence of SEQ IDNO: 515, connected to the fifth sequence of SEQ ID NO: 32. See FIG. 83for sequences.

In some embodiments, the CoStAR (and/or fusion protein) comprises theMSLN scFv of one or more of the following: SS1, MN5, HN1, M912, huTP218,or P4 (e.g., as depicted in FIG. 84 ). In some embodiments, the linker,spacer, transmembrane domain, and intracellular domain are shared acrossdifferent CoStARs. In some embodiments, changing the MSLN scFv does notaffect expression of the CoStAR.

In some embodiments the CoStAR (and/or fusion protein) comprises a MSLNtargeting scFv. In some embodiments the MSLN targeting CoStAR (and/orfusion protein) comprises signal peptide, a scFv and an intracellularsignaling domain. In some embodiments the domains of the MSLN targetingCoStAR (and/or fusion protein) are connected via linker sequences. Insome embodiments the MSLN targeting CoStAR (and/or fusion protein)comprises a hOncostatin-M signal peptide and an intracellular CD28-CD40domain. In some embodiments the MSLN targeting domain comprises any ofSS1, M5, HN1, M912, huTP218, or P4 scFvs, comprising SEQ ID NO: 186-191in FIG. 84 . In some embodiments the MSLN targeting CoStAR (and/orfusion protein) further comprises a linker with the sequence AAAGSGbetween the scFv and spacer. In some embodiments the spacer is a CD28ECspacer. In some embodiments the MSLN targeting CoStAR (and/or fusionprotein) further comprises a CD28 transmembrane domain.

In some embodiments, the MSLN targeting CoStAR (and/or fusion protein)is comprised of a sequence with 75% identity to the polypeptide in SEQID NO: 192, 210, 228, 246, 264, or 282 in FIG. 84 . In some embodiments,the MSLN targeting CoStAR (and/or fusion protein) is comprised of asequence with 80% identity to the polypeptide in SEQ ID NO: 192, 210,228, 246, 264, or 282 in FIG. 84 . In some embodiments, the MSLNtargeting CoStAR (and/or fusion protein) is comprised of a sequence with85% identity to the polypeptide in SEQ ID NO: 192, 210, 228, 246, 264,or 282 in FIG. 84 . In some embodiments, the MSLN targeting CoStAR(and/or fusion protein) is comprised of a sequence with 90% identity tothe polypeptide in SEQ ID NO: 192, 210, 228, 246, 264, or 282 in FIG. 84. In some embodiments, the MSLN targeting CoStAR (and/or fusion protein)is comprised of a sequence with 95% identity to the polypeptide in SEQID NO: 192, 210, 228, 246, 264, or 282 in FIG. 84 . In some embodiments,the MSLN targeting CoStAR (and/or fusion protein) in SEQ ID NO: 192,210, 228, 246, 264, or 282 in FIG. 84 has 1 point mutation. In someembodiments, the MSLN targeting CoStAR (and/or fusion protein) in SEQ IDNO: 192, 210, 228, 246, 264, or 282 in FIG. 84 has 2 point mutations. Insome embodiments, the MSLN targeting CoStAR (and/or fusion protein) inSEQ ID NO: 192, 210, 228, 246, 264, or 282 in FIG. 84 has 3, 4, or 5point mutations.

In some embodiments, the MSLN targeting domain of the constructcomprises a SS1, M5, HN1, M912, huTP218, or P4 scFv, comprising asequence with at least 75% identity to the polypeptide in SEQ ID NO:186-191 in FIG. 84 . In some embodiments, the MSLN targeting domaincomprises a SS1, M5, HN1, M912, huTP218, or P4 scFv, comprising asequence with at least 80% identity to the polypeptide in SEQ ID NO:186-191 in FIG. 84 . In some embodiments, the MSLN targeting domaincomprises a SS1, M5, HN1, M912, huTP218, or P4 scFv, comprising asequence with at least 85% identity to the polypeptide in SEQ ID NO:186-191 in FIG. 84 . In some embodiments, the MSLN targeting domaincomprises a SS1, M5, HN1, M912, huTP218, or P4 scFv, comprising asequence with at least 90% identity to the polypeptide in SEQ ID NO:186-191 in FIG. 84 . In some embodiments, the MSLN targeting domaincomprises a SS1, M5, HN1, M912, huTP218, or P4 scFv, comprising asequence with at least 95% identity to the polypeptide in SEQ ID NO:186-191 in FIG. 84 . In some embodiments, the MSLN targeting scFV in SEQID NO: 186-191 in FIG. 84 has 1 point mutation. In some embodiments, theMSLN targeting scFV in SEQ ID NO: 186-191 in FIG. 84 has 2 pointmutations. In some embodiments, the MSLN targeting scFV in SEQ ID NO:186-191 in FIG. 84 has 3, 4, or 5 point mutations.

In some embodiments the CoStAR (and/or fusion protein) comprises aCEA.ICOS.CD40 arrangement. In some embodiments the CEA.ICOS.CD40 CoStAR(and/or fusion protein) comprises signal peptide, a scFv and anintracellular signaling domain. In some embodiments the domains of theCEA.ICOS.CD40 CoStAR (and/or fusion protein) are connected via linkersequences. In some embodiments the CEA.ICOS.CD40 CoStAR (and/or fusionprotein) comprises a hOncostatin-M signal peptide, humanized MFE23 VHand VL, human ICOS Q9Y6W8, and a human intracellular CD40 domain. Insome embodiments the CEA.ICOS.CD40 CoStAR (and/or fusion protein)further comprises a 3xG4S linker between the VH and VL. In someembodiments the CEA.ICOS.CD40 CoStAR (and/or fusion protein) furthercomprises a linker with the sequence AAAGSGGSG between the VL and ICOSdomain.

In some embodiments, any of the sequence components in any of thefigures and/or tables (such as table 7) can be combined into any of theCoStAR arrangements provided herein (such as a binding domain, TM, ICOSor CD28 domain, and CD40 domain (with other optional spacers and/orlinkers). In some embodiments, any of the sequence components in any ofthe figures and/or tables (such as table 7) can be combined into any ofthe fusion protein arrangements provided herein. Such fusion proteinscan include the amino acid sequences as provided herein. In someembodiments, the CoStARs and/or fusion proteins are at least 70, 75, 80,85, 90, 95, 96, 97, 98, 99, or 100% identical to any one ore more of theamino acid sequences provided herein, including any range between anytwo of the preceeding values.

In some embodiments, the CEA.ICOS.CD40 CoStAR (and/or fusion protein) iscomprised of a sequence with at least 75% identity to the polypeptide inSEQ ID NO: 348 in FIG. 83 . In some embodiments, the CEA.ICOS.CD40CoStAR (and/or fusion protein) is comprised of a sequence with at least80% identity to the polypeptide in SEQ ID NO: 348 in FIG. 83 . In someembodiments, the CEA.ICOS.CD40 CoStAR (and/or fusion protein) iscomprised of a sequence with at least 85% identity to the polypeptide inSEQ ID NO: 348 in FIG. 83 . In some embodiments, the CEA.ICOS.CD40CoStAR (and/or fusion protein) is comprised of a sequence with at least90% identity to the polypeptide in SEQ ID NO: 348 in FIG. 83 . In someembodiments, the CEA.ICOS.CD40 CoStAR (and/or fusion protein) iscomprised of a sequence with at least 95% identity to the polypeptide inSEQ ID NO: 348 in FIG. 83 . In some embodiments, the CEA.ICOS.CD40CoStAR (and/or fusion protein) in SEQ ID NO: 348 in FIG. 83 has 1 pointmutation. In some embodiments, the CEA.ICOS.CD40 CoStAR (and/or fusionprotein) in SEQ ID NO: 348 in FIG. 83 has 2 point mutations. In someembodiments, the CEA.ICOS.CD40 CoStAR (and/or fusion protein) in SEQ IDNO: 348 in FIG. 83 has 3, 4, or 5 point mutations.

In some embodiments, the CEA targeting domain comprises a scFv,comprising a sequence with at least 75% identity to the polypeptide inSEQ ID NO: 12 in FIG. 83 . In some embodiments, the CEA targeting domaincomprises a scFv, comprising a sequence with at least 80% identity tothe polypeptide in SEQ ID NO: 12 in FIG. 83 . In some embodiments, theCEA targeting domain comprises a scFv, comprising a sequence with atleast 85% identity to the polypeptide in SEQ ID NO: 12 in FIG. 83 . Insome embodiments, the CEA targeting domain comprises a scFv, comprisinga sequence with at least 90% identity to the polypeptide in SEQ ID NO:12 in FIG. 83 . In some embodiments, the CEA targeting domain comprisesa scFv, comprising a sequence with at least 95% identity to thepolypeptide in SEQ ID NO: 12 in FIG. 83 . In some embodiments, the CEAtargeting scFV in SEQ ID NO: 12 in FIG. 83 has 1 point mutation. In someembodiments, the CEA targeting scFV in SEQ ID NO: 12 in FIG. 83 has 2point mutations. In some embodiments, the CEA targeting scFV in SEQ IDNO: 12 in FIG. 83 has 3, 4, or 5 point mutations.

In some embodiments, the fusion protein comprises: a binding domainspecific for CEA linked to; a transmembrane domain that is linked to; anICOS domain that is linked to; a CD40 signaling domain.

In some embodiments, the fusion protein comprises: a binding domainspecific for MSLN linked to; a transmembrane domain that is linked to; aCD28 domain that is linked to; a CD40 signaling domain.

In some embodiments, the fusion protein comprises: a first sequence thatis at least 70% identical to SEQ ID NO: 12; a second sequence that is atransmembrane domain; a third sequence that is at least 70% identical toSEQ ID NO: 518; and a fourth sequence that is at least 70% identical toSEQ ID NO: 32.

In some embodiments, the fusion protein comprises: a first sequence thatis at least 70% identical to any one of SEQ ID NO: 186-191; a secondsequence that is a transmembrane domain; a third sequence that is atleast 70% identical to SEQ ID NO: 25; and a fourth sequence that is atleast 70% identical to SEQ ID NO: 32.

In some embodiments, the fusion protein comprises: a HCDR1 that is anHCDR1 in SEQ ID NO: 12; a HCDR2 that is an HCDR2 in SEQ ID NO: 12; aHCDR3 that is an HCDR3 in SEQ ID NO: 12; a LCDR1 that is an LCDR1 in SEQID NO: 12; a LCDR2 that is an LCDR2 in SEQ ID NO: 12; a LCDR3 that is anHCDR3 in SEQ ID NO: 12, wherein 1, 2, 3, 4, 5, or 6 of the LCDRs caninclude 1, 2, or 3 point mutations; a second sequence that is atransmembrane domain; a third sequence that is at least 70% identical toSEQ ID NO: 515; and a fourth sequence that is at least 70% identical toSEQ ID NO: 32.

In some embodiments, the fusion protein further comprises a signalpeptide sequence that is at least 70% identical to SEQ ID NO: 1.

In some embodiments, the fusion protein further comprises a linkersequence that is at least 70% identical to SEQ ID NO: 18.

In some embodiments, the fusion protein further comprises an ICOSsequence that is at least 70% identical to SEQ ID NO: 515.

In some embodiments, the fusion protein further comprises an CD40sequence that is at least 70% identical to SEQ ID NO: 32.

In some embodiments, the fusion protein comprises: a HCDR1 that is anHCDR1 in SEQ ID NOs: 186-191; a HCDR2 that is an HCDR2 in SEQ ID NOs:186-191; a HCDR3 that is an HCDR3 in SEQ ID NOs: 186-191; a LCDR1 thatis an LCDR1 in SEQ ID NOs: 186-191; a LCDR2 that is an LCDR2 in SEQ IDNOs: 186-191; a LCDR3 that is an HCDR3 in SEQ ID NOs: 186-191, wherein1, 2, 3, 4, 5, or 6 of the LCDRs can include 1, 2, or 3 point mutations;a second sequence that is a transmembrane domain; a third sequence thatis at least 70% identical to SEQ ID NO: 25; and a fourth sequence thatis at least 70% identical to SEQ ID NO: 32.

In some embodiments, the fusion protein further comprises an CD28 TMsequence that is at least 70% identical to SEQ ID NO: 19.

In some embodiments, the fusion protein further comprises an CD28sequence that is at least 70% identical to SEQ ID NO: 25.

As used herein, “full length protein” or “full length receptor” refersto a receptor protein, such as, for example, a CD28 receptor protein.The term “full length” encompasses receptor proteins lacking up to about5 or up to 10 amino acids, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10amino acids, at the N-terminal of the mature receptor protein once itssignal peptide has been cleaved. For instance, while a specific cleavagesite of a receptors N-terminal signal peptide may be defined,variability in exact point of cleavage has been observed. The term “fulllength” does not imply presence or absence of amino acids of thereceptors N-terminal signal peptide. In one embodiment, the term “fulllength” (e.g. a full length CD28 or a full length CD40 intracellulardomain, according to some embodiments) encompasses mature receptorproteins (e.g. CD28 according to some embodiments) lacking the Nterminal signal peptide lacking up to about 5, for example 1, 2, 3, 4,5, or up to 10 amino acids at the N-terminal of the mature receptorprotein once its signal peptide has been cleaved. As mentioned above, a“full length” CD28 receptor or other receptor or tumor antigen bindingdomain according to some embodiments does not include the signal peptideand may lack up to about 5, for example 1, 2, 3, 4, 5, or up to 10 aminoacids at the N-terminal of the mature receptor protein (e.g. N terminalresidues N, K, I, L and/or V). This is shown in the exemplary fusions,e.g. SEQ ID Nos. 433-441 (note that these may lack up to about 5, forexample 1, 2, 3, 4, 5, or up to 10 amino acids at the N-terminal of themature receptor protein as shown in the boxed region).

CoStARs have modular form and can be constructed to compriseextracellular, transmembrane and intracellular domains obtained from aone or more proteins, along with the scFv obtained from an antibody thatbinds to a disease-associated antigen, for example, a tumor associatedantigen.

In some embodiments, a CoStAR comprises a disease-associated, forexample a tumor-associated, antigen receptor, such as but not limited toa tumor-associated antigen specific scFv, and a primary costimulatoryreceptor protein that is capable of binding to its cognate ligand andproviding an intracellular signal. In some embodiments, the primarycostimulatory receptor can be less than a full length protein but issufficient to bind cognate ligand and transduce a signal. In someembodiments, the primary costimulatory receptor domain is full length,such as but not limited to, full length CD28. Thus, both the antigenspecific binding domain and the ligand specific receptor are capable ofbinding cognate antigen and ligand respectively. The amino acidsequences provided herein provide embodiments of several CoStARconstructs. These include CoStARs constructs that comprise an antigenbinding domain, an optional spacer, an optional costimulatory receptorprotein comprising an extracellular ligand binding segment or fragmentthereof and intracellular CD40 signaling domain. In another embodiment,a CoStAR comprises an antigen binding domain, an optional spacer, anextracellular ligand-binding portion of a costimulatory receptorprotein, a transmembrane domain, and an intracellular signaling domainof a selected costimulatory receptor protein and intracellular CD40signaling domain. In some embodiments, the extracellular ligand-bindingportion comprises a CD28 truncation, for example, a C-terminal CD28truncation after amino acids IEV, and is followed by an intracellularsignaling domain. In some embodiments, the intracellular signalingdomain is from CD40. The transmembrane domain separating theextracellular ligand-binding and intracellular signaling domains can befrom, with limitation, CD28, CD40. In further embodiments, CoStARs cancomprise additional costimulatory domains, for example a third,intracellular costimulatory signaling domain and in this respect may besimilar to certain chimeric antigen receptors (CARs), which have beenclassified into first (CD3ζ only), second (one costimulatorydomain+CD3ζ), or third generation (more than one costimulatorydomain+CD3ζ).

Costimulatory receptor proteins useful in CoStARs can include, withoutlimitation, CD2, CD9, CD26, CD27, CD28, CD29, CD38, CD40, CD43, CD46,CD49d, CD55, CD73, CD81, CD82, CD99, CD100, CD134 (OX40), CD137 (41BB),CD150 (SLAM), CD270 (HVEM), CD278 (ICOS), CD357 (GITR), or EphB6, whichin their natural form comprise extracellular ligand binding domains andintracellular signal transducing domains. For example, CD2 ischaracterized as a cell adhesion molecule found on the surface of Tcells and is capable of initiating intracellular signals necessary for Tcell activation. CD27 is characterized as a type II transmembraneglycoprotein belonging to the TNFR superfamily (TNFRSF) whose expressionon B cells is induced by antigen-receptor activation in B cells. CD28 isone of the proteins on T cells and is the receptor for CD80 (B7.1) andCD86 (B7.2) ligands on antigen-presenting cells. CD137 (4-1BB) ligand isfound on most leukocytes and on some non-immune cells. OX40 ligand isexpressed on many antigen-presenting cells such as DC2s (dendriticcells), macrophages, and B lymphocytes. In one embodiment, thecostimulatory receptor protein is full length CD28 as defined herein.

CD40 is a member of the tumor necrosis factor receptor (TNFR)superfamily and several isoforms are generated by alternative splicing.Its ligand, CD154 (also called CD40L) is a protein that is primarilyexpressed on activated T cells. For reference, the human CD40 isoform 1protein sequence is set forth in GenBank accession No. NP_001241.1,including signal peptide (amino acids 1-20), transmembrane domain (aminoacids 194-215), and cytoplasmic domain (amino acids 216-277)(SEQ IDNO:32). CD40 receptor signaling involves adaptor proteins including butnot limited to TNF receptor-associated factors (TRAF), and the CD40cytoplasmic domain comprises signaling components, including amino acidsequences fitting an SH3 motif (KPTNKAPH) (SEQ ID NO:35), TRAF2 motif(PKQE (SEQ ID NO:36), PVQE (SEQ ID NO:37), SVQE (SEQ ID NO:38)), TRAF6motif (QEPQEINFP) (SEQ ID NO:39) and PKA motif (KKPTNKA (SEQ ID NO:40),SRISVQE (SEQ ID NO:41)). Some embodiments include engineered signalingdomains, such as engineered CD40 signaling domains, comprisingTRAF-binding amino acid sequences. Engineered signaling domains thatbind to TRAF1, TRAF2, TRAF3, and TRAF5 may comprise the major consensussequence (P/S/A/T)X(Q/E)E or minor consensus sequence PXQXXD and can beidentified in or obtained from, without limitation, TNFR family memberssuch as CD30, OX40, 41BB, and the EBV oncoprotein LMP1. (See, e.g., Ye,H et al., The Structural Basis for the Recognition of Diverse ReceptorSequences by TRAF2. Molecular Cell, 1999; 4(3):321-30. doi:10.1016/51097-2765(00)80334-2; Park H H, Structure of TRAF Family:Current Understanding of Receptor Recognition. Front. Immunol. 2018;9:1999. doi: 10.3389/fimmu.2018.01999; Chung, J. Y. et al., All TRAFsare not created equal: common and distinct molecular mechanisms ofTRAF-mediated signal transduction. Journal of Cell Science 2002;115:679-688).

Examples disclosed herein demonstrate operation of CD40 as acostimulatory signaling domain in a CoStAR and further that cytokine andchemokine expression profiles are altered by signaling domain selection.In some embodiments, the costimulatory CD40 signaling domain of a CoStARpromotes pro-inflammatory cytokines (e.g., IL-2, TNFα). In someembodiments, the costimulatory CD40 signaling domain of a CoStAR reducesimmunosuppressive cytokines (e.g., IL-5, IL-10). Costimulatory activityof a CD40 signaling domain or fragment can be observed in combinationwith a first receptor signaling domain such as but not limited to CD2,CD9, CD26, CD27, CD28, CD29, CD38, CD40, CD43, CD46, CD49d, CD55, CD73,CD81, CD82, CD99, CD100, CD134 (OX40), CD137 (41BB), CD150 (SLAM), CD270(HVEM), CD278 (ICOS), CD357 (GITR), or EphB6, as compared to activity ofthe first receptor signaling domain without the CD40 signaling domain orfragment. In this regard, the CD40 signaling domains, includingsignaling fragments comprising particular factor binding sites orwherein particular factor binding sites are mutated, in combination witha costimulatory first signaling domain, are capable of promoting orsuppressing relative expression of particular cytokines and/orchemokines as compared to the first signaling domain alone. activity ofa costimulatory signaling domain. (See, e.g., Ahonen, C L et al., TheCD40-TRAF6 axis controls affinity maturation and the generation oflong-lived plasma cells. Nat Immunol. 2002; 3: 451-456; Mackey M F etal., Distinct contributions of different CD40 TRAF binding sites toCD154-induced dendritic cell maturation and IL-12 secretion. Eur JImmunol. 2003; 33: 779-789; Mukundan L et al., TNF receptor-associatedfactor 6 is an essential mediator of CD40-activated proinflammatorypathways in monocytes and macrophages. J Immunol. 2005; 174: 1081-1090.

In some embodiments, a CoStAR comprises substantially all of a CD40costimulatory domain. In some embodiments, a CoStAR comprises two ormore CD40 costimulatory domains. In some embodiments, a CoStAR comprisesa CD40 costimulatory domain signaling component or fragment or motif. Insome embodiments, the CD40 signaling fragment or motif comprises,consists, or consists essentially of an SH3 binding sequence (e.g.,without limitation, KPTNKAPH (SEQ ID NO:35), PTNKAPHP (SEQ ID NO:443) orPTNKAPH (SEQ ID NO:444)), TRAF2/TRAF3 binding sequence (e.g., withoutlimitation, PKQE (SEQ ID NO:506), PKQET (SEQ ID NO:445), PVQE (SEQ IDNO:507), PVQET (SEQ ID NO:446), SVQE (SEQ ID NO:508), SVQET (SEQ IDNO:447)), TRAF6 binding sequence (e.g., without limitation, PQEINF (SEQID NO:509), QEPQEINF (SEQ ID NO:448) or QEPQEINFP (SEQ ID NO:39)) or PKAsequence (e.g., without limitation, KKPTNKA (SEQ ID NO:40), or SRISVQE(SEQ ID NO:41) as well as two or more, or three or more, or four or moresuch components or motifs, or combinations thereof, which can be inmultiple copies and arranged in any order. In some embodiments, a CoStARcomprises a CD40 costimulatory domain and a CD40 costimulatory domainsignaling component or motif. In some embodiments, one or more of theSH3, TRAF2/TRAF3, TRAF6, or PKA motifs of the CD40 signaling domain ismutated. In some embodiments, the SH3 motif, TRAF2/TRAF3 motif, andTRAF6 motif are sufficient to modulate pro-inflammatory and/orimmunosuppressive cytokines. In some embodiments, adding tandem copiesof those motifs and/or mutating certain motifs amplifies these effects.

TABLE 1 provides non-limiting examples of TRAF2/TRAF3 binding sequences.Exemplary TRAF2/TRAF3 binding sequences SEQ ID Source P-4 P-3 P-2 P-1 P0P1 P2 P3 P4 P5 P6 NO. hTNFR2 P F S K E E C A F R S 450 hCD40 A A P V Q ET L H G C 451 hCD30 M L S V E E E G K E D 452 hCD27 T I P I Q E D Y R KP 453 hLTβR S T P H Q E D G K A W 454 hATAR T V A V E E T I P S T 455hOX40 R T P I Q E E Q A D A 456 m41BB T G A A Q E E D A C S 457 m41BB RC P Q E E E G G G G 458 h41BB V Q T T Q E E D G C S 459 h41BB R F P E EE E G G C E 460 bLMP1 R T P V Q E S G Y P D 461 bLMP1 R P P V Q E T G GG G 462 bLMP1 H P P V Q E T G G G G 463 bLMP1 H P P V Q E T G E G G 464bLMP1 H P P I Q E T G N G G 465 LAT A L S S Q E A E E V E 466 hTANK S VP I Q C T D K T D 467 hLMP1 P H P Q Q A T D D S S 468 rLMP1 P Y P I Q AT D G G N 469 rLMP1 P H P I Q A T D G A N 470 rLMP1 P Y P V Q A S D G GD 471 Minor P/S/ X Q/E E 472 Consensus A/T Major P V Q E 473 Consensus

Accordingly, TRAF2/TRAF3 binding sequences of some embodiments furtherinclude sequences such as P₁V₂Q₃E₄ and variants wherein P₁ issubstituted with S, A, or T, V₂ is substituted with Q, K, or E, Q₃ issubstituted with E, and/or E₄ is substituted with A. In such variants,any one, two, three, or all four of P₁V₂Q₃E₄ may be substituted.Non-limiting examples are shown in Table 1 at positions P−2, P−1, P0,P1.

Illustrative non-limiting examples of CD40 TRAF2/TRAF3 sequence variantsinclude the following, the amino acids at P−2, P−1, P0, and P1 enclosedby dashes, and the TRAF2/TRAF3 source protein identified.

TABLE 2 Exemplary CD40 TRAF2/TRAF3 variants SEQ TRAF2/3 IDAmino acid sequence sequece NO: based on CD40 origin 474KKVAKKPTNKAPHPKQEPQEI CD40 WT NFPDDLPGSNTAA--PVQE--TLHGCQPVTQEDGKESRI--S VQE--RQ 475 KKVAKKPTNKAPHPKQEPQEI CD40/NFPDDLPGSNTAA--TQEE-- v41BB TLHGCQPVTQEDGKESRISVQ ERQ 476KKVAKKPTNKAPHPKQEPQEI CD40/ NFPDDLPGSNTAA--SKEE-- VTNFR2TLHGCQPVTQEDGKESRISVQ ERQ 477 KKVAKKPTNKAPHPKQEPQEI CD40/NFPDDLPGSNTAA--AVEE-- vATAR TLHGCQPVTQEDGKESRISVQ ERQ 478KKVAKKPTNKAPHPKQEPQEI CD40/ NFPDDLPGSNTAAPVQETLHG vATARCQPVTQEDGKESRI--AVEE- -RQ 479 KKVAKKPTNKAPHPKQEPQEI CD40/NFPDDLPGSNTAAPVQETLHG v41BB CQPVTQEDGKESRI--PEEE- -RQ

TABLE 3 provides non-limiting examples of TRAF2/TRAF3 binding sequences.Exemplary TRAF6 binding sequences SEQ ID P-2 P-1 P0 P1 P2 P3 NO: hCD40 PQ E I N F 480 hTRANCE-R P Q E I D F 481 Mal P P E L R F 482 TRIF P E E MS W 483 IRAK (1) P Q E N S Y 484 IRAK (2) P V E S D E 485 IRAK (3) P E ES D E 486 IRAK-2 (1) P E E T D E 487 IRAK-2 (2) P T E N G E 488 IRAK-M PV E D D E 489 RIP2 P P E N Y E 490 MyD88 P S E L R F 491 Consensus P X EX X Ac/Ar 492 Ac = acidic residue; Ar = Aromatic residue

Illustrative non-limiting examples of CD40 TRAF6 sequence variantsinclude the following, the amino acids at P−2, P−1, P0, P1, P2, and P3enclosed by dashes, and the TRAF6 sequence origin identified.

TABLE 4 Exemplary CD40 TRAF6 variants SEQ TRAF6 ID Amino acid sequencesequece No: based on CD40 origin 493 KKVAKKPTNKAPHPKQE--PQEIN CD40 WTF--PDDLPGSNTAAPVQE TLHGCQPVTQEDGKESRISVQERQ 494 KKVAKKPTNKAPHPKQE--PEEMSCD40/ W--PDDLPGSNTAAPVQE TLHGCQPVTQEDGKESRISVQERQ vTRIF 495KKVAKKPTNKAPHPKQE--PPENY CD40/ E--PDDLPGSNTAAPVQETLHGCQPVTQEDGKESRISVQERQ vRIP2 496 KKVAKKPTNKAPHPKQE--PQENS CD40/Y--PDDLPGSNTAAPVQE TLHGCQPVTQEDGKESRISVQERQ vIRAK(1)

TABLE 5  Exemplary CD40 TRAF2/TRAF3/TRAF6 consensus variants SEQ IDAmino acid sequence No: variants based on CD40 497KKVAKKPTNKAPHPKQE--PQEI CD40 NF--PDDLPGSNTAA--PVQE-- WTTLHGCQPVTQEDGKESRI--SVQ E--RQ 498 KKVAKKPTNKAPHPKQE--PXEX X(Ac/Ar)--PDDLPGSNTAAPVQETLHGCQPV TQEDGKESRISVQERQ 499 KKVAKKPTNKAPHPKQEPQEINFPDDLPGSNTAA-- (P/S/A/T)X(Q/E)E--TLHGC QPVTQEDGKESRISVQERQ 500KKVAKKPTNKAPHPKQEPQEINF PDDLPGSNTAAPVQE TLHGCQPVTQEDGKESRI--(P/S/A/T)X(Q/E)E--RQ 501 KKVAKKPTNKAPHPKQE--PXEX X(Ac/Ar)--PDDLPGSNTAA--(P/S/A/T)X(Q/E)--ETLHGC QPVTQEDGKESRISVQERQ 502 KKVAKKPTNKAPHPKQEPQEINFPDDLPGSNTAA--(P/S/A/T)X (Q/E)E--TLHGCQPVTQEDGKE SRI-- (P/S/A/T) X (Q/E)E--RQ 503 KKVAKKPTNKAPHPKQE--PXEX X(Ac/Ar)--PDDLPGSNTAAPVQETLHGCQPVTQEDGKESRI--(P/ S/A/T)X(Q/E)E--RQ 504 KKVAKKPTNKAPHPKQE PXEXX(Ac/Ar)--PDDLPGSNTAA-- (P/S/A/T)X(Q/E)E--TLHGC QPVTQEDGKESRI--(P/S/A/T) X (Q/E)E--RQ 505 KKVAKKPTNKAPHPKQE--PEELRW--PDDLPGSNTAA--THEE-- TLHGCQPVTQEDGKESRI--AIEE --RQ Ar = aromaticresidue; Ac = acidic residue

In some embodiments, selection of one or more costimulatory domainsignaling component or motif is guided by the cell in which the CoStARis to be expressed and/or a desired costimulatory activity more closelyidentified with a signaling component or motif, or avoidance of acostimulatory activity more closely identified with a signalingcomponent or motif.

In some embodiments, a CoStAR signaling domain comprises, in addition toa CD40 costimulatory domain or signaling component or motif thereof, ortwo or more such domains or components or motifs or combinationsthereof, an additional full length costimulatory domain or signalingcomponent thereof from, without limitation, CD2, CD9, CD26, CD27, CD28,CD29, CD38, CD40, CD43, CD46, CD49d, CD55, CD73, CD81, CD82, CD99,CD100, CD134 (OX40), CD137 (41BB), CD150 (SLAM), CD270 (HVEM), CD278(ICOS), CD357 (GITR), or EphB6.

For reference, the human CD28 protein sequence is set forth in GenBankaccession No. NP_006130.1, including signal peptide (amino acids 1-18),extracellular domain (amino acids 19-152), transmembrane domain (aminoacids 153-179) and cytoplasmic domain (amino acids 180-200). Theextracellular domain includes an immunoglobulin type domain (amino acids21-136) which contains amino acids with compose the antigen binding siteand amino acids that form the homodimer interface. The extracellulardomain includes several asparagine residues which may be glycosylated,and the intracellular domain comprises serine and tyrosine residues,which may be phosphorylated.

In some embodiments, the fusion protein comprises a CD40 signalingdomain linked to the CD28 signaling domain. The CD40 signaling domaincan provide co-stimulatory signal to the cell upon recognition of MSLNby the scFV. In some embodiments, the co-stimulatory signal provided bythe CD40 signaling domain can enhance cell survival and proliferation.The co-stimulatory signal provided from the CD28 and CD40 signalingdomains upon MSLN recognition by the binding domain can be sufficient topromote survival and proliferation of fusion protein expressing cells,the CD40 signaling domain can comprise SEQ ID NO: 521. In someembodiments, the CD40 signaling domain can comprise an SH3 motif, TRAF2motif (SEQ ID NO:516, 517, or 518), TRAF6 motif (SEQ ID NO: 519), PKA(SEQ ID NO: 520 or 521), or a combination thereof, where the sequencelist is shown in Table 7. In some embodiments, the CD40 domain is simplythe amino acid structure shown in SEQ ID NO: 521, or one at least 60,70, 80, 90, 95, 96, 97, 98, 99, or 100% identical thereto.

For reference, the human CD8 alpha chain protein sequence is set forthby GenBank accession No. NP_001139345.1, including signal peptide (aminoacids 1-21), extracellular domain (amino acids 22-182), transmembranedomain (amino acids 183-203), and cytoplasmic domain (amino acids204-235). The extracellular domain includes an immunoglobulin typedomain (amino acids 28-128) which contains amino acids with compose theantigen binding site and amino acids that form the homodimer interface.The extracellular domain includes several asparagine residues which maybe glycosylated, and the intracellular domain comprises serine andtyrosine residues, which may be phosphorylated.

For reference, the human IgG4 constant region sequence is set forth inUniProtKB/Swiss-Prot: accession No. P01861.1, including CH1 (amino acids1-98), hinge (amino acids 99-110), CH2 (amino acids 111-220), CH3 (aminoacids 221-327). The CH2 region includes asparagine at amino acid 177,which is the glycosylated and associated with Fc receptor andantibody-dependent cell-mediated cytotoxicity (ADCC).

For reference, the protein sequence of human CD137 (4-1BB), another TNFRsuperfamily member, is set forth by GenBank accession No. NP_001552.2,including signal peptide (amino acids 1-23), extracellular domain (aminoacids 24-186), transmembrane domain (amino acids 187-213), andcytoplasmic domain (amino acids 214-255). Binding of CD137L ligandtrimers expressed on antigen presenting cells to CD137 leads to receptortrimerization and activation of signaling cascades involved in T cellreactivity and survival (Li et al., Limited Cross-Linking of 4-1BB by4-1BB Ligand and the Agonist Monoclonal Antibody Utomilumab. CellReports 2018; 25:909-920). Coimmunoprecipitation of CD137 with thesignaling adaptors TRAF-2 and TRAF-1 and the structural basis for theinteractions has been reported (Ye, H et al., Molecular Cell, 1999;4(3):321-30).

For reference, the human CD134 (OX40) protein sequence is set forth byGenBank accession No. NP_003318.1, including signal peptide (amino acids1-28), extracellular domain (amino acids 29-214), transmembrane domain(amino acids 215-235), and cytoplasmic domain (amino acids 236-277).This receptor has been shown to activate NF-kappaB through itsinteraction with adaptor proteins TRAF2 and TRAF5 and studies suggestthat this receptor promotes expression of apoptosis inhibitors BCL2 andBCL21L1/BCL2-XL.

The human T-cell surface antigen CD2 has at least two isoforms. Forreference, the human CD2 isoform1 protein sequence is set forth byNP_001315538.1, including signal peptide (amino acids 1-24),extracellular domain (amino acids 25-235), transmembrane domain (aminoacids 236-261), and cytoplasmic domain (amino acids 262-377). The humanCD2 isoform2 protein sequence is set forth by NP_001758.2

For reference, the human CD357 (GITR) isoform-1 protein sequence is setforth by GenBank accession No. NP_004186.1, including signal peptide(amino acids 1-25), extracellular domain (amino acids 26-162),transmembrane domain (amino acids 163-183), and cytoplasmic domain(amino acids 184-241).

For reference, the human CD29 (beta1 integrin) protein sequence is setforth by GenBank accession No. NP_596867, including signal peptide(amino acids 1-20), extracellular domain (amino acids 21-728),transmembrane domain (amino acids 729-751), and cytoplasmic domain(amino acids 752-798).

The human CD150 (SLAM) protein sequence has at several isoforms. Inaddition to the transmembrane form of CD150 (mCD150), cells ofhematopoietic lineage express mRNA encoding the secreted form of CD150(sCD150), which lacks the entire transmembrane region of 30 amino acids.For reference, human SLAM isoform b is set forth by GenBank accessionNo. NP_003028.1, including signal peptide (amino acids 1-20),extracellular domain (amino acids 21-237), transmembrane domain (aminoacids 238-258), and cytoplasmic domain (amino acids 259-335). Human SLAMisoform a is set forth by GenBank accession No. NP_001317683.1.

CD278 or ICOS (Inducible T cell COStimulator) is a CD28-superfamilycostimulatory molecule that is expressed on activated T cells. HumanICOS precursor (199 aa with signal peptide) is set forth by GenBankaccession No. NP_036224.1, including signal peptide (amino acids 1-20),an Ig-V-like domain (amino acids 21-140), transmembrane domain (aminoacids 141-161) and intracellular domain (amino acids 162-199). ICOScontains an IProx motif sequence SSSVHDPNGE (SEQ ID NO:466). The IProxmotif sequence SSSXXXPXGE (SEQ ID NO:467) resembles certain bindingsites of TRAF1 (SASFQRPQSE (SEQ ID NO:468)), TRAF2 (SSSFQRPVND (SEQ IDNO:469)), TRAF3 (SSFKKPTGE (SEQ ID NO:470)), and TRAF5 (SSSFKRPDGE (SEQID NO:471)).

Hematopoietic cell signal transducer (HCST) also known as DAP10, KAP10,PIK3AP, and hematopoietic cell signal transducer (GenBank accession No.NP_055081.1) encodes a transmembrane signaling adaptor thought to formpart of a receptor complex with the C-type lectin-like receptor NKG2D.The intracellular domain contains a YxxM motif of a phosphatidylinositol3-kinase binding site.

In some embodiments the CoStAR targets carcinoembryonic antigen (CEA).In some embodiments, the CoStAR targets mesothelin (MSLN).

In some embodiments, the CEA targeting CoStAR targets Carcinoembryonicantigen-related cell adhesion molecule 5 (CEACAM5) NCBI accession:NM_001291484.3. In some embodiments, the CEA scFv binds to CEACAM5.

In some embodiments, a CoStAR may be expressed alone under the controlof a promoter in a therapeutic population of cells that have therapeuticactivity, for example, Tumour Infiltrating Lymphocytes (TILs).Alternatively, the CoStAR may be expressed along with a therapeutictransgene such as a chimeric antigen receptor (CAR) and/or T-cellReceptor (TCR), (note that may lack up to about 5, for example 1, 2, 3,4, 5, or up to 10 amino acids at the N-terminal of the mature receptorprotein). Thus, some embodiments also relate to CoStAR constructs, notlimited to those having a sequence as shown in any of SEQ ID NOS:42-185,192-335, 344-430, including one of these sequences which lacks up toabout 5, for example 1, 2, 3, 4, 5, or up to 10 amino acids at theN-terminal of the mature receptor protein). Suitable TCRs and CARs arewell known in the literature, for example HLA-A*02-NYESO-1 specific TCRs(Rapoport et al. Nat Med 2015) or anti-CD19scFv.CD3ζ fusion CARs(Kochenderfer et al. J Clin Oncol 2015) which have been successfullyused to treat Myeloma or B-cell malignancies respectively. The CoStARsdescribed herein may be expressed with any known CAR or TCR thusproviding the cell with a regulatable growth switch to allow cellexpansion in-vitro or in-vivo, and a conventional activation mechanismin the form of the TCR or CAR for anti-cancer activity. Thus someembodiments provide a cell for use in adoptive cell therapy comprising aCoStAR as described herein and a TCR and/or CAR that specifically bindsto a tumor associated antigen. An exemplary CoStAR comprising CD28includes an extracellular antigen binding domain and an extracellular,transmembrane and intracellular signaling domain.

The term “antigen binding domain” as used herein refers to an antibodyfragment including, but not limited to, a diabody, a Fab, a Fab′, aF(ab′)2, an Fv fragment, a disulfide stabilized Fv fragment (dsFv), a(dsFv)2, a bispecific dsFv (dsFv-dsFv′), a disulfide stabilized diabody(ds diabody), a single-chain antibody molecule (scFv), an scFv dimer(bivalent diabody), a multispecific antibody formed from a portion of anantibody comprising one or more CDRs, a camelized single domainantibody, a nanobody, a domain antibody, a bivalent domain antibody, orany other antibody fragment that binds to an antigen but does notcomprise a complete antibody structure. An antigen binding domain iscapable of binding to the same antigen to which the parent antibody or aparent antibody fragment (e.g., a parent scFv) binds. In someembodiments, an antigen-binding fragment may comprise one or morecomplementarity determining regions (CDRs) from a particular humanantibody grafted to frameworks (FRs) from one or more different humanantibodies. An “antigen binding domain” may be referred to as a “ligandbinding domain.”

In some embodiments, the scFV comprises a VH and/or VL with at 70%identity to the polypeptides in SEQ ID NOs: 12. In some embodiments, thescFV comprises a VH and/or VL with at 75% identity to the polypeptidesin SEQ ID NOs: 12. In some embodiments, the scFV comprises a VH and/orVL with at 80% identity to the polypeptides in SEQ ID NOs: 12. In someembodiments, the scFV comprises a VH and/or VL with at 85% identity tothe polypeptides in SEQ ID NOs: 12. In some embodiments, the scFVcomprises a VH and/or VL with at 90% identity to the polypeptides in SEQID NOs: 12. In some embodiments, the CDRs of SEQ ID NOs: 12 have 1 pointmutation. In some embodiments, the CDRs of SEQ ID NOs: 12 have 2 pointmutations. In some embodiments, the CDRs of SEQ ID NOs: 12 have 3, 4 or5 point mutations. In some embodiments, the sequence(s) are those shownin FIG. 83 . In some embodiments, the binding domain is defined by theamino acid structure alone, and can be any one of those sequencesprovided herein regarding such amino acid structures. It shall beappreciated that all embodiments disclosed herein regarding CEA alsoapply for the corresponding arrangements in Table 7.

In some embodiments, any of the structural arrangements in FIG. 28 canbe used with any of the sequences in Table 7, or any of the other tablesherein, and/or any of the noted sequences or related sequences in thefigures. In some embodiments, any of HDR1, HCDR2, HCDR3, LCDR1, LCDR2,LCDR3, VH, VL, and/or the scFV disclosed in Table 7, or any of the othertables herein, and/or any of the noted sequences and/or relatedsequences in the figures can be used in one or more of the arrangementsin FIG. 28 . In some embodiments, any of the scFv sequences, especiallythe CEA scFv identified sequences can be used in this manner. In someembodiments, any of the above sequences, or any sequences that are atleast 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical orsimilar thereto can be used in FIG. 28 . In some embodiment, the CoStARcan have components that are functionally characterized orcharacterizable as provided herein. In some embodiment, the fusionprotein can have components that are purely structurally defined (suchas by amino acid or nucleic acid sequence) as provided herein. In someembodiments, a fusion protein is provided that includes an amino acidsequence that is at least 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or100% identical or similar to SEQ ID NO: 348. In some embodiments, anyone or more of the sequences in FIG. 83 can be used in an arrangementFIG. 28 , including any sequence that is at least 70, 75, 80, 85, 90,95, 96, 97, 98, 99, or 100% identical or similar to any one or more thesequences.

In some embodiments, the scFV comprises a VH and/or VL with at 70%identity to the polypeptides in any one of SEQ ID NOs: 186-191. In someembodiments, the scFV comprises a VH and/or VL with at 75% identity tothe polypeptides in any one of SEQ ID NOs: 186-191. In some embodiments,the scFV comprises a VH and/or VL with at 80% identity to thepolypeptides in any one of SEQ ID NOs: 186-191. In some embodiments, thescFV comprises a VH and/or VL with at 85% identity to the polypeptidesin any one of SEQ ID NOs: 186-191. In some embodiments, the scFVcomprises a VH and/or VL with at 90% identity to the polypeptides in anyone of SEQ ID NOs: 186-191. In some embodiments, the CDRs of any one ofSEQ ID NOs: 186-191 have 1 point mutation. In some embodiments, the CDRsof any one of SEQ ID NOs: 186-191 have 2 point mutations. In someembodiments, the CDRs of any one of SEQ ID NOs: 186-191 have 3, 4 or 5point mutations. In some embodiments, the sequence(s) are those shown inFIG. 84 . In some embodiments, the binding domain is defined by theamino acid structure alone, and can be any one of those sequencesprovided herein regarding such amino acid structures. It shall beappreciated that all embodiments disclosed herein regarding MSLN alsoapply for the corresponding arrangements in Table 7.

In some embodiments, any of the structural arrangements in any one ormore of FIGS. 62, 63A, 64, 65, 66, 67, 69B, 70A, 70B, and 84A can beused with any of the sequences in Table 7, or any of the other tablesherein, and/or any of the noted sequences or related sequences in thefigures. In some embodiments, any of HDR1, HCDR2, HCDR3, LCDR1, LCDR2,LCDR3, VH, VL, and/or the scFV disclosed in Table 7, or any of the othertables herein, and/or any of the noted sequences and/or relatedsequences in the figures can be used in one or more of the arrangementsin any one or more of FIGS. 62, 63A, 64, 65, 66, 67, 69B, 70A, 70B, and84A. In some embodiments, any of the scFv sequences, especially the CEAscFv identified sequences can be used in this manner. In someembodiments, any of the above sequences, or any sequences that are atleast 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical orsimilar thereto can be used in any one or more of FIGS. 62, 63A, 64, 65,66, 67, 69B, 70B, and 84A. In some embodiment, the CoStAR can havecomponents that are functionally characterized or characterizable asprovided herein. In some embodiment, the fusion protein can havecomponents that are purely structurally defined (such as by amino acidor nucleic acid sequence) as provided herein. In some embodiments, afusion protein is provided that includes an amino acid sequence that isat least 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical orsimilar to any one or more of SEQ ID NOs: 192, 210, 228, 246, 264,and/or 282. In some embodiments, any one or more of the sequences inFIGS. 84A-84C can be used in an arrangement of any one or more of FIGS.62, 63A, 64, 65, 66, 67, 69B, 70B, and 84A, including any sequence thatis at least 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical orsimilar to any one or more the sequences.

The antigen binding domain can be made specific for anydisease-associated antigen, including but not limited totumor-associated antigens (TAAs) and infectious disease-associatedantigens. In some embodiments, the ligand binding domain is bispecific.Antigens have been identified in most of the human cancers, includingBurkitt lymphoma, neuroblastoma, melanoma, osteosarcoma, renal cellcarcinoma, breast cancer, prostate cancer, lung carcinoma, and coloncancer. TAA's include, without limitation, CD19, CD20, CD22, CD24, CD33,CD38, CD123, CD228, CD138, BCMA, GPC3, CEA, folate receptor (FRα),mesothelin, CD276, gp100, 5T4, GD2, EGFR, MUC-1, PSMA, EpCAM, MCSP,SM5-1, MICA, MICB, ULBP and HER-2. TAAs further include neoantigens,peptide/MHC complexes, and HSP/peptide complexes.

In some embodiments, the antigen binding domain comprises a T-cellreceptor or binding fragment thereof that binds to a defined tumorspecific peptide-MHC complex. The term “T cell receptor,” or “TCR,”refers to a heterodimeric receptor composed of αβ or γδ chains that pairon the surface of a T cell. Each α, β, γ, and δ chain is composed of twoIg-like domains: a variable domain (V) that confers antigen recognitionthrough the complementarity determining regions (CDR), followed by aconstant domain (C) that is anchored to cell membrane by a connectingpeptide and a transmembrane (TM) region. The TM region associates withthe invariant subunits of the CD3 signaling apparatus. Each of the Vdomains has three CDRs. These CDRs interact with a complex between anantigenic peptide bound to a protein encoded by the majorhistocompatibility complex (pMHC) (Davis and Bjorkman (1988) Nature,334, 395-402; Davis et al. (1998) Annu Rev Immunol, 16, 523-544; Murphy(2012), xix, 868 p.).

In some embodiments, the antigen binding domain comprises a naturalligand of a tumor expressed protein or tumor-binding fragment thereof. Anon-limiting example is PD1 which binds to PDL1. Another example is thetransferrin receptor 1 (TfR1), also known as CD71, a homodimeric proteinthat is a key regulator of cellular iron homeostasis and proliferation.Although TfR1 is expressed at a low level in a broad variety of cells,it is expressed at higher levels in rapidly proliferating cells,including malignant cells in which overexpression has been associatedwith poor prognosis. In some embodiments, the antigen binding domaincomprises transferrin or a transferrin receptor-binding fragmentthereof.

In some embodiments, the antigen binding domain is specific to a definedtumor associated antigen, such as but not limited to FRα, CEA, 5T4,CA125, SM5-1 or CD71. In some embodiments, the tumor associated antigencan be a tumor-specific peptide-MHC complex. In certain suchembodiments, the peptide is a neoantigen. In other embodiments, thetumor associated antigen it a peptide-heat shock protein complex.

In some embodiments, the binding domain allows targeting of the cancertreatment specifically to CEA or MSLN expressing cancer cells. In someembodiments, the binding domain can comprise an scFv, a peptide, anantibody heavy-chain, a natural ligand, or a receptor specific for CEAor MSLN. In some embodiments, the binding domain can comprise apolypeptide comprising an scFv with the sequence comprising any one ofSEQ ID NO: 186-191, where the sequence is shown in FIG. 84 . In someembodiments, the binding domain can be linked to the transmembranedomain by a linker and/or a spacer. In some embodiments, the bindingdomain is that in SEQ ID NO: 185-191. In some embodiments, the bindingdomain is at least 70, 80, 90, 95, 96, 97, 98, 99 or 100% identical tothat in SEQ ID NO: 186-191 (or any of the corresponding sequences for adifferent target in FIG. 84 ). In some embodiments, the binding domaincomprises a VH and/or VL that is at least 70, 80, 90, 95, 96, 97, 98, 99or 100% identical to the VH, and/or VL in SEQ ID NOs: 12 (or any of thecorresponding sequences for a different target in FIG. 83 , such as forMSLN or CEA). In some embodiments, the binding domain comprises a HCDR1,HCDR2, HCDR3, LCDR1, LCDR2, and/or LCDR3 that is at least 70, 80, 90, or100% identical to the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and/or LCDR3 inSEQ ID NOs: 186-191 and 12 (or any of the corresponding sequences for adifferent target in FIG. 83 or 84 ).

In some embodiments, a CoStAR comprises:

i. an scFv that binds to carcinoembryonic antigen (CEA) or mesothelin(MSLN), a spacer and transmembrane sequence of CD28, an ICOS domain or aCD28 signaling domain, and a CD40 signaling domain.

ii. an scFv that binds to CEA or MSLN, a spacer and transmembranesequence of CD28, and a CD40 signaling domain.

iii. an scFv that binds to CEA or MSLN, a spacer and transmembranesequence of CD28, a CD137 signaling domain, and a CD40 signaling domain.

iv. an scFv that binds to CEA or MSLN, a spacer and transmembranesequence of CD28, a CD134 signaling domain, and a CD40 signaling domain.

v. an scFv that binds to CEA or MSLN, a spacer and transmembranesequence of CD28, a CD2 signaling domain, and a CD40 signaling domain.

vi. an scFv that binds to CEA or MSLN, a spacer and transmembranesequence of CD28, a GITR signaling domain, and a CD40 signaling domain.

vii. an scFv that binds to CEA or MSLN, a spacer and transmembranesequence of CD28, a CD29 signaling domain, and a CD40 signaling domain.

viii. an scFv that binds to CEA or MSLN, a spacer and transmembranesequence of CD28, a CD150 signaling domain, and a CD40 signaling domain.

ix. an scFv that binds to CEA or MSLN, a spacer and transmembranesequence of CD8, a CD28 signaling domain, and a CD40 signaling domain.

x. an scFv that binds to CEA or MSLN, a spacer and transmembranesequence of CD8, and a CD40 signaling domain.

xi. an scFv that binds to CEA or MSLN, a spacer and transmembranesequence of CD8, a CD137 signaling domain, and a CD40 signaling domain.

xii. an scFv that binds to CEA or MSLN, a spacer and transmembranesequence of CD8, a CD134 signaling domain, and a CD40 signaling domain.

xiii. an scFv that binds to CEA or MSLN, a spacer and transmembranesequence of CD8, a CD2 signaling domain, and a CD40 signaling domain.

xiv. an scFv that binds to CEA or MSLN, a spacer and transmembranesequence of CD8, a GITR signaling domain, and a CD40 signaling domain.

xv. an scFv that binds to CEA or MSLN, a spacer and transmembranesequence of CD8, a CD29 signaling domain, and a CD40 signaling domain.

xvi. an scFv that binds to CEA or MSLN, a spacer and transmembranesequence of CD8, a CD150 signaling domain, and a CD40 signaling domain.

xvii. an scFv that binds to CEA or MSLN, a spacer comprising an IgG4constant region and CD28 transmembrane sequence, a CD28 signalingdomain, and a CD40 signaling domain.

xviii. an scFv that binds to CEA or MSLN, a spacer comprising an IgG4constant region and CD28 transmembrane sequence, and a CD40 signalingdomain.

xix. an scFv that binds to CEA or MSLN, a spacer comprising an IgG4constant region and CD28 transmembrane sequence, a CD137 signalingdomain, and a CD40 signaling domain.

xx. an scFv that binds to CEA or MSLN, a spacer comprising an IgG4constant region and CD28 transmembrane sequence, a CD134 signalingdomain, and a CD40 signaling domain.

xxi. an scFv that binds to CEA or MSLN, a spacer comprising an IgG4constant region and CD28 transmembrane sequence, a CD2 signaling domain,and a CD40 signaling domain.

xxii. an scFv that binds to CEA or MSLN, a spacer comprising an IgG4constant region and CD28 transmembrane sequence, a GITR signalingdomain, and a CD40 signaling domain.

xxiii. an scFv that binds to CEA or MSLN, a spacer comprising an IgG4constant region and CD28 transmembrane sequence, a CD29 signalingdomain, and a CD40 signaling domain.

xxiv. an scFv that binds to CEA or MSLN, a spacer comprising an IgG4constant region and CD28 transmembrane sequence, a CD150 signalingdomain, and a CD40 signaling domain.

xxv. an scFv that binds to CEA or MSLN, a spacer comprising an IgG4constant region and CD28 transmembrane sequence, a first CD40 signalingdomain and a second CD40 signaling domain

xxvi. an scFv that binds to CEA or MSLN, a spacer comprising an IgG4constant region and CD28 transmembrane sequence, a first CD40 signalingdomain and a second mutated CD40 signaling domain

xxvii. an scFv that binds to CEA or MSLN, a binding domain that binds toPDL1, a short spacer and transmembrane sequence of CD28, a CD28signaling domain, and a CD40 signaling domain.

xxviii. an scFv that binds to CEA or MSLN, a binding domain that bindsto PDL1, a short spacer and transmembrane sequence of CD28, and a CD40signaling domain.

xxix. an scFv that binds to CEA or MSLN, a binding domain that binds toCD155, CD112, or CD113, a CD28 transmembrane domain, a CD28 signalingdomain, and a CD40 signaling domain.

xxx. a binding domain that binds to CD155, CD112, or CD113, a CD28transmembrane domain, and a CD40 signaling domain.

xxxi. an scFv that binds to CEA or MSLN, a binding domain that binds toPDL1, a short spacer and transmembrane sequence of CD28, a CD28signaling domain, and a CD40 signaling domain.

xxxii. an scFv that binds to CEA or MSLN, a binding domain that binds toPDL1, a short spacer and transmembrane sequence of CD28, and a CD40signaling domain.

xxxiii. an scFv that binds to CEA or MSLN, a binding domain that bindsto CD155, CD112, or CD113, a short spacer and transmembrane sequence ofCD28, a CD28 signaling domain, and a CD40 signaling domain.

xxxiv. an scFv that binds to CEA or MSLN, a binding domain that binds toCD155, CD112, or CD113, a short spacer and transmembrane sequence ofCD28, and a CD40 signaling domain.

xxxv. an scFv that binds to CEA or MSLN, a spacer comprising the CD28extracellular domain, and transmembrane sequence of CD28, and a NTRK1signaling domain

xxxvi. an scFv that binds to CEA or MSLN, a spacer comprising the CD28extracellular domain and transmembrane sequence of CD28, a NTRK1signaling domain, and a CD40 signaling domain.

xxxvii. an scFv that binds to CEA or MSLN, a spacer comprising the CD28extracellular domain and transmembrane sequence of CD28, a CD28signaling domain, and a NTRK1 signaling domain.

xxxviii. an scFv that binds to CEA or MSLN, a spacer comprising the CD28extracellular domain and transmembrane sequence of CD28, a CD28signaling domain, a NTRK1 signaling domain, and a CD40 signaling domain.

xxxix. an scFv that binds to CEA or MSLN, a spacer comprising the ICOSextracellular domain and transmembrane sequence of ICOS, an ICOSsignaling domain, and a CD40 signaling domain

xl. an scFv that binds to CEA or MSLN, a spacer comprising the CD28extracellular domain and transmembrane sequence of CD28, a CD28signaling domain, and an ICOS signaling domain.

xli. an scFv that binds to CEA or MSLN, a spacer comprising the CD28extracellular domain and transmembrane sequence of CD28, a CD28signaling domain, an ICOS signaling domain, and a CD40 signaling domain.

xlii. an scFv that binds to CEA or MSLN, a spacer comprising the CD2extracellular domain and transmembrane sequence of CD2, a CD2 signalingdomain, and a CD40 signaling domain.

xliii. an scFv that binds to CEA or MSLN, a spacer comprising the CD28extracellular domain and transmembrane sequence of CD28, a CD28signaling domain, and a CD2 signaling domain.

xliv. an scFv that binds to CEA or MSLN, a spacer comprising the CD28extracellular domain and transmembrane sequence of CD28, a CD28signaling domain, a CD40 signaling domain, and a CD2 signaling domain.

xlv. an scFv that binds to CEA or MSLN, a spacer comprising the CD28extracellular domain and transmembrane sequence of CD28, a CD28signaling domain, and a CD137 signaling domain.

xlvi. an scFv that binds to CEA or MSLN, a spacer comprising the CD28extracellular domain and transmembrane sequence of CD28, a CD28signaling domain, a CD40 signaling domains, and a CD137 signalingdomain.

xlvii. an scFv that binds to CEA or MSLN, a spacer comprising the CD28extracellular domain and transmembrane sequence of CD28, a CD28signaling domain, and a DAP10 signaling domain.

xlviii. an scFv that binds to CEA or MSLN, a spacer comprising the CD28extracellular domain and transmembrane sequence of CD28, a CD28signaling domain, a CD40 signaling domain, and a DAP10 signaling domain.

xlix. an scFv that binds to CEA or MSLN, a spacer comprising the CD28extracellular domain and transmembrane sequence of CD28, a CD28signaling domain, and a CD134 signaling domain.

xlx. an scFv that binds to CEA or MSLN, a spacer comprising the CD28extracellular domain and transmembrane sequence of CD28, a CD40signaling domain, and a CD134 signaling domain.

In some embodiments, a CoStAR comprises an scFv that binds to MSLNlinked to the spacer, transmembrane, and signaling domain structure ofany one of paragraphs i-xlx.

In some embodiments, a CoStAR comprises an scFv that binds to FolR1linked to the spacer, transmembrane, and signaling domain structure ofany one of paragraphs i-xlx.

In some embodiments, a CoStAR comprises the spacer, transmembrane, andsignaling domain structure of any one of i-xxxiv and binds to FolR1 orMSLN by a binding domain which comprises an antigen-binding fragment ofscFv MOV19 (SEQ ID NO:9).

In some embodiments, a CoStAR comprises the spacer, transmembrane, andsignaling domain structure of any one of i-xxxiv and binds to CEA by abinding domain which comprises an antigen-binding fragment of scFv MFE23(SEQ ID NO:10).

In some embodiments, a CoStAR comprises the spacer, transmembrane, andsignaling domain structure of any one of i-xxvi and xxxi to xxxiv andbinds to CEA by a binding domain which comprises an antigen-bindingfragment of scFv MFE23(K>Q) (SEQ ID NO:11).

In some embodiments, a CoStAR comprises the spacer, transmembrane, andsignaling domain structure of any one of i-xxvi and xxxi to xxxiv andbinds to CEA by a binding domain which comprises an antigen-bindingfragment of humanized scFv MFE23 (hMFE23) (SEQ ID NO:12).

In some embodiments, a CoStAR comprises the spacer, transmembrane, andsignaling domain structure of any one of i-xxvi and xxxi to xxxiv andbinds to CEA by a binding domain which comprises an antigen-bindingfragment of scFv CEA6 (SEQ ID NO:13).

In some embodiments, a CoStAR comprises the spacer, transmembrane, andsignaling domain structure of any one of i-xxvi and xxxi to xxxiv andbinds to CEA by a binding domain which comprises an antigen-bindingfragment of scFv BW431/26 (SEQ ID NO:14).

In some embodiments, a CoStAR comprises the spacer, transmembrane, andsignaling domain structure of any one of i-xxvi and xxxi to xxxiv andbinds to CEA by a binding domain which comprises an antigen-bindingfragment of scFv HuT84.66(M5A) (SEQ ID NO:15).

In some embodiments, a CoStAR comprises the spacer, transmembrane, andsignaling domain structure of any one of i-xxxiv and binds to FolR1 by abinding domain which comprises an antigen-binding fragment of scFv MOV19(SEQ ID NO:9).

In some embodiments, a CoStAR comprises the spacer, transmembrane, andsignaling domain structure of any one of i-xxxiv and xxxi to xxxiv andbinds to MSLN by a binding domain which comprises an antigen-bindingfragment of scFv SS1 (SEQ ID NO:186).

In some embodiments, a CoStAR comprises the spacer, transmembrane, andsignaling domain structure of any one of i-xxvi and xxxi to xxxiv andbinds to MSLN by a binding domain which comprises an antigen-bindingfragment of scFv M5 (humanized SS1) (SEQ ID NO:187).

In some embodiments, a CoStAR comprises the spacer, transmembrane, andsignaling domain structure of any one of i-xxvi and xxxi to xxxiv andbinds to MSLN by a binding domain which comprises an antigen-bindingfragment of humanized scFv HN1 (SEQ ID NO:188).

In some embodiments, a CoStAR comprises the spacer, transmembrane, andsignaling domain structure of any one of i-xxvi and xxxi to xxxiv andbinds to MSLN by a binding domain which comprises an antigen-bindingfragment of scFv M912 (SEQ ID NO:189 or SEQ ID NO:511).

In some embodiments, a CoStAR comprises the spacer, transmembrane, andsignaling domain structure of any one of i-xxvi and xxxi to xxxiv andbinds to MSLN by a binding domain which comprises an antigen-bindingfragment of scFv HuYP218 (SEQ ID NO:190 or SEQ ID NO: 512).

In some embodiments, a CoStAR comprises the spacer, transmembrane, andsignaling domain structure of any one of i-xxvi and xxxi to xxxiv andbinds to MSLN by a binding domain which comprises an antigen-bindingfragment of scFv P4 (SEQ ID NO:191 or SEQ ID NO: 513).

As use herein, the term “specifically binds” or “is specific for” refersto measurable and reproducible interactions, such as binding between atarget and an antibody or antibody moiety that is determinative of thepresence of the target in the presence of a heterogeneous population ofmolecules, including biological molecules. For example, an antibodymoiety that specifically binds to a target (which can be an epitope) isan antibody moiety that binds the target with greater affinity, avidity,more readily, and/or with greater duration than its bindings to othertargets. In some embodiments, an antibody moiety that specifically bindsto an antigen reacts with one or more antigenic determinants of theantigen (for example a cell surface antigen or a peptide/MHC proteincomplex) with a binding affinity that is at least about 10 times itsbinding affinity for other targets. In some embodiments, specificallybinds denotes an affinity of at least 10⁶, 10⁷, 10⁸, 10⁹, or 10¹⁰ M⁻¹.Specific binding is detectably higher in magnitude and distinguishablefrom non-specific binding occurring to at least one unrelated target.Specific binding can be the result of formation of bonds betweenparticular functional groups or particular spatial fit (e.g., lock andkey type) whereas nonspecific binding is usually the result of van derWaals forces. Specific binding does not however necessarily imply thatan antibody or fusion protein binds one and only one target.

A full length antibody structural unit is a tetramer of subunits. Eachtetramer includes two identical pairs of polypeptide chains, each pairhaving one “light” (about 25 kDa) and one “heavy” chain (about 50-70kDa). The amino-terminal portion of each chain includes a variableregion of about 100 to 110 or more amino acids primarily responsible forantigen recognition. This variable region is initially expressed linkedto a cleavable signal peptide. The variable region without the signalpeptide is sometimes referred to as a mature variable region. Thus, forexample, a light chain mature variable region means a light chainvariable region without the light chain signal peptide. However,reference to a variable region does not mean that a signal sequence isnecessarily present; and in fact signal sequences are cleaved once theantibodies or fusion proteins have been expressed and secreted. A pairof heavy and light chain variable regions defines a binding region of anantibody. The carboxy-terminal portion of the light and heavy chainsrespectively defines light and heavy chain constant regions.

Light chains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, and define theantibody's isotype as IgG, IgM, IgA, IgD and IgE, respectively. Withinlight and heavy chains, the variable and constant regions are joined bya “J” segment of about 12 or more amino acids, with the heavy chain alsoincluding a “D” segment of about 10 or more amino acids. (See generally,Fundamental Immunology (Paul, W., ed., 2nd ed. Raven Press, N.Y., 1989),Ch. 7) (incorporated by reference in its entirety for all purposes).

The mature variable regions of each light/heavy chain pair form theantibody binding site. Thus, an intact antibody has two binding sites,i.e., is divalent. In natural antibodies, the binding sites are thesame. However, bispecific antibodies can be made in which the twobinding sites are different (see, e.g., Songsivilai S, Lachmann P C.1990. Bispecific antibody: a tool for diagnosis and treatment ofdisease. Clin Exp Immunol. 79:315-321; Kostelny S A, Cole M S, Tso J Y.1992. Formation of bispecific antibody by the use of leucine zippers. JImmunol. 148: 1547-1553). The variable regions all exhibit the samegeneral structure of relatively conserved framework regions (FR) joinedby three hypervariable regions, also called complementarity determiningregions or CDRs. The CDRs from the two chains of each pair are alignedby the framework regions, enabling binding to a specific epitope. FromN-terminal to C-terminal, both light and heavy chains comprise thedomains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. For convenience, thevariable heavy CDRs can be referred to as HCDR1, HCDR2 and HCDR3; thevariable light chain CDRs can be referred to as LCDR1, LCDR2 and LCDR3.The assignment of amino acids to each domain is in accordance with thedefinitions of Kabat E A, et al. 1987 and 1991. Sequences of Proteins ofImmunological Interest (National Institutes of Health, Bethesda, Md.) orChothia C, Lesk A M. 1987. Canonical Structures for the HypervariableRegions of Immunoglobulins. J Mol Biol 196:901-917; Chothia C, et al.1989. Conformations of Immunoglobulin Hypervariable Regions. Nature342:877-883. Kabat also provides a widely used numbering convention(Kabat numbering) in which corresponding residues between differentheavy chain variable regions or between different light chain variableregions are assigned the same number. Although Kabat numbering can beused for antibody constant regions, EU numbering can also be used.

The term “epitope” refers to a site on an antigen to which an antibodybinds. An epitope on a protein can be formed from contiguous amino acidsor noncontiguous amino acids juxtaposed by tertiary folding of one ormore proteins.

As noted herein, the binding of the CoStAR is achieved via a bindingdomain, which can include an antibody or binding fragment thereof.Examples of binding fragments include Fv, Fab′, Fab′-SH, F(ab′)2;diabodies; linear antibodies; single-chain antibody molecules (e.g.scFv); and multispecific antibodies formed from antibody fragments. scFvantibodies are described in Houston J S. 1991. Methods in Enzymol.203:46-96. In addition, antibody fragments comprise single chainpolypeptides having the characteristics of a VH domain, namely beingable to assemble together with a VL domain, or of a VL domain, namelybeing able to assemble together with a VH domain to a functional antigenbinding site and thereby providing the antigen binding property of fulllength antibodies.

Spacer

In some embodiments, a CoStAR optionally comprises a spacer regionbetween the antigen binding domain and the costimulatory receptor. Asused herein, the term “spacer” refers to the extracellular structuralregion of a CoStAR that separates the antigen binding domain from theexternal ligand binding domain of the costimulatory protein. The spacerprovides flexibility to access the targeted antigen and receptor ligand.In some embodiments long spacers are employed, for example to targetmembrane-proximal epitopes or glycosylated antigens (see Guest R. D. etal. The role of extracellular spacer regions in the optimal design ofchimeric immune receptors: evaluation of four different scFvs andantigens. J. Immunother. 2005; 28:203-211; Wilkie S. et al., Retargetingof human T cells to tumor-associated MUC1: the evolution of a chimericantigen receptor. J. Immunol. 2008; 180:4901-4909). In otherembodiments, CoStARs bear short spacers, for example to target membranedistal epitopes (see Hudecek M. et al., Receptor affinity andextracellular domain modifications affect tumor recognition byROR1-specific chimeric antigen receptor T cells. Clin. Cancer Res. 2013;19:3153-3164; Hudecek M. et al., The nonsignalling extracellular spacerdomain of chimeric antigen receptors is decisive for in vivo antitumoractivity. Cancer Immunol. Res. 2015; 3:125-135). In some embodiments,the spacer comprises all or part of or is derived from an IgG hinge,including but not limited to IgG1, IgG2, or IgG4. By “derived from an Ighinge” is meant a spacer comprising insertions, deletions, or mutationsin an IgG hinge. In some embodiments, a spacer can comprise all or partof one or more antibody constant domains, such as but not limited to CH2and/or CH3 domains. In some embodiments, in a spacer comprising all orpart of a CH2 domain, the CH2 domain is modified so as not to bind to anFc receptor. For example, Fc receptor binding in myeloid cells has beenfound to impair CAR T cell functionality. In some embodiments, thespacer comprises all or part of an Ig-like hinge from CD28, CD8, orother protein comprising a hinge region. In some embodiments, the CoStARcomprises a spacer, the spacer is from 1 and 50 amino acids in length.

In an non-limiting embodiment, the spacer comprises essentially all ofan extracellular domain, for example a CD28 extracellular domain (i.e.from about amino acid 19, 20, 21, or 22 to about amino acid 152) or anextracellular domain of another protein, including but not limited toanother TNFR superfamily member. In an embodiment, the spacer comprisesa portion of an extracellular domain, for example a portion of a CD28extracellular domain, and may lack all or most of the Ig domain. Inanother embodiment, the spacer includes amino acids of CD28 from about141 to about 152 but not other portions of the CD28 extracellulardomain. In another embodiment, the spacer includes amino acids of CD8from about 128 to about 182 but not other portions of the CD8extracellular domain.

In some embodiments, the spacer in an anti-MSLN CoStAR is a CD28 spacerdomain, truncated CD28 spacer domain, or CD8 spacer domain. In someembodiments, changing the spacer domain can result in increasedsecretion of T cell effector cytokines upon recognition of signal 1 andsignal 2. In some embodiments the T cell effector cytokines are IL-2,IFNγ, and TNFα.

Linker

In some embodiments, the CoStAR extracellular domain comprises a linker.Linkers comprise short runs of amino acids used to connect domains, forexample a binding domain with a spacer or transmembrane domain. In orderfor there to be flexibility to bind ligand, a ligand binding domain willusually be connected to a spacer or a transmembrane domain by flexiblelinker comprising from about 5 to 25 amino amino acids, such as, forexample, AAAGSGGSG (SEQ ID NO:18), GGGGSGGGGSGGGGS (SEQ ID NO:431). Insome embodiments, a CoStAR comprises a binding domain joined directly toa transmembrane domain by a linker, and without a spacer. In someembodiments, a CoStAR comprises a binding domain joined directly to atransmembrane by a spacer and without a linker.

Signaling Domain

As discussed above, in some embodiments, a CoStAR comprises a fulllength primary costimulatory receptor which can comprise anextracellular ligand binding and intracellular signaling portion of,without limitation, CD2, CD9, CD26, CD27, CD28, CD29, CD38, CD40, CD43,CD46, CD49d, CD55, CD73, CD81, CD82, CD99, CD100, CD134 (OX40), CD137(41BB), CD150 (SLAM), CD270 (HVEM), CD278 (ICOS), CD357 (GITR), orEphB6. In other embodiments, the costimulatory receptor comprises achimeric protein, for instance comprising an extracellular ligandbinding domain of one of the aforementioned proteins and anintracellular signaling domain of another of the aforementionedproteins. In some embodiments, the signaling portion of the CoStARcomprises a single signaling domain. In other embodiments, the signalingportion of the CoStAR comprises a second intracellular signaling domainsuch as but not limited to: CD2, CD27, CD28, CD40, CD134 (OX40), CD137(4-1BB), CD150 (SLAM). In some embodiments, the first and secondintracellular signaling domains are the same. In other embodiments, thefirst and second intracellular signaling domains are different. In someembodiments, the costimulatory receptor is capable of dimerization.Without being bound by theory, it is thought that CoStARs dimerize orassociate with other accessory molecules for signal initiation. In someembodiments, CoStARs dimerize or associate with accessory moleculesthrough transmembrane domain interactions. In some embodiments,dimerization or association with accessory molecules is assisted bycostimulatory receptor interactions in the intracellular portion, and/orthe extracellular portion of the costimulatory receptor.

Transmembrane Domain

Although the main function of the transmembrane is to anchor the CoStARin the T cell membrane, in some embodiments, the transmembrane domaininfluences CoStAR function. In some embodiments, the transmembranedomain is comprised by the full length primary costimulatory receptordomain. In embodiments, the CoStAR construct comprises an extracellulardomain of one receptor and an intracellular signaling domain of a secondreceptor, the transmembrane domain can be that of the extracellulardomain or the intracellular domain. In some embodiments, thetransmembrane domain is from CD4, CD8a, CD28, or ICOS. Gueden et al.associated use of the ICOS transmembrane domain with increased CAR Tcell persistence and overall anti-tumor efficacy (Guedan S. et al.,Enhancing CAR T cell persistence through ICOS and 4-1BB costimulation.JCI Insight. 2018; 3:96976). In an embodiment, the transmembrane domaincomprises a hydrophobic α helix that spans the cell membrane.

In an embodiment, the transmembrane domain comprises amino acids of theCD28 transmembrane domain from about amino acid 153 to about amino acid179. In another embodiment, the transmembrane domain comprises aminoacids of the CD8 transmembrane domain from about amino acid 183 to aboutamino acid 203. In some embodiments, the CoStARs may include severalamino acids between the transmembrane domain and signaling domain. Forexample, in one construct described herein the link from a CD8transmembrane domain to a signaling domain comprises several amino acidsof the CD8 cytoplasmic domain (e.g., amino acids 204-210 of CD8).

Variants

In some embodiments, amino acid sequence variants of the antibodymoieties or other moieties provided herein are contemplated. Forexample, it may be desirable to improve the binding affinity and/orother biological properties of the antibody moiety. Amino acid sequencevariants of an antibody moiety may be prepared by introducingappropriate modifications into the nucleotide sequence encoding theantibody moiety, or by peptide synthesis. Such modifications include,for example, deletions from, and/or insertions into and/or substitutionsof residues within the amino acid sequences of the antibody moiety. Anycombination of deletion, insertion, and substitution can be made toarrive at the final construct, provided that the final constructpossesses the desired characteristics, e.g., antigen-binding.

In some embodiments, antibody binding domain moieties comprising one ormore amino acid substitutions, deletions, or insertions are provided.Sites of interest for mutational changes include the antibody bindingdomain heavy and light chain variable regions (VRs) and frameworks(FRs). Amino acid substitutions may be introduced into a binding domainof interest and the products screened for a desired activity, e.g.,retained/improved antigen binding or decreased immunogenicity. In someembodiments, amino acid substitutions may be introduced into one or moreof the primary co-stimulatory receptor domain (extracellular orintracellular), secondary costimulatory receptor domain, orextracellular co-receptor domain. Accordingly, some embodimentsencompass CoStAR and/or fusion proteins and component parts particularlydisclosed herein as well as variants thereof, i.e. CoStAR and/or fusionproteins and component parts having at least 75%, at least 80%, at least85%, at least 87%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99% sequence identity to the amino acid sequencesparticularly disclosed herein. The terms “percent similarity,” “percentidentity,” and “percent homology” when referring to a particularsequence are used as set forth in the University of Wisconsin GCGsoftware program BestFit. Other algorithms may be used, e.g. BLAST,psiBLAST or TBLASTN (which use the method of Altschul et al. (1990) J.Mol. Biol. 215: 405-410), FASTA (which uses the method of Pearson andLipman (1988) PNAS USA 85: 2444-2448).

Particular amino acid sequence variants may differ from a referencesequence by insertion, addition, substitution or deletion of 1 aminoacid, 2, 3, 4, 5-10, 10-20 or 20-30 amino acids. In some embodiments, avariant sequence may comprise the reference sequence with 1, 2, 3, 4, 5,6, 7, 8, 9, 10 or more residues inserted, deleted or substituted. Forexample, 5, 10, 15, up to 20, up to 30 or up to 40 residues may beinserted, deleted or substituted.

In some preferred embodiments, a variant may differ from a referencesequence by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more conservativesubstitutions. Conservative substitutions involve the replacement of anamino acid with a different amino acid having similar properties. Forexample, an aliphatic residue may be replaced by another aliphaticresidue, a non-polar residue may be replaced by another non-polarresidue, an acidic residue may be replaced by another acidic residue, abasic residue may be replaced by another basic residue, a polar residuemay be replaced by another polar residue or an aromatic residue may bereplaced by another aromatic residue. Conservative substitutions may,for example, be between amino acids within the following groups:

Conservative substitutions are shown in Table 6 below.

TABLE 6 Amino Acid Substitutions Original Preferred Residue ExemplarySubstitutions Substitutions Ala (A) Val; Leu; Ile Val Arg (R) Lys; Gln;Asn Lys Asn (N) Gln; His; Asp; Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C)Ser; Ala Ser Gln (Q) Asn; Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala AlaHis (H) Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val; Met; Ala; Phe;Norleucine Leu Leu (L) Norleucinne; Ile; Val; Met; Ala; Phe Ile Lys (K)Arg; Gln; Asn Arg Met (M) Leu; Phe; Ile Leu Phe (F) Trp; Leu; Val; Ile;Ala; Tyr Tyr Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Val; Ser Ser Trp(W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr; Ser Phe Val (V) Ile; Leu; Met;Phe; Ala; Norleucine Leu

Amino acids may be grouped into different classes according to commonside-chain properties: a. hydrophobic: Norleucine, Met, Ala, Val, Leu,Ile; b. neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; c. acidic: Asp,Glu; d. basic: His, Lys, Arg; e. residues that influence chainorientation: Gly, Pro; aromatic: Trp, Tyr, Phe. Non-conservativesubstitutions will entail exchanging a member of one of these classesfor another class.

Cells

The cells may be any lymphocyte that is useful in adoptive cell therapy,such as a T-cell or a natural killer (NK) cell, an NKT cell, agamma/delta T-cell or T regulatory cell. The cells may be allogeneic orautologous to the patient.

T cells or T lymphocytes are a type of lymphocyte that have a centralrole in cell-mediated immunity. They can be distinguished from otherlymphocytes, such as B cells and natural killer cells (NK cells), by thepresence of a T-cell receptor (TCR) on the cell surface. There arevarious types of T cell, as summarised below. Cytotoxic T cells (TCcells, or CTLs) destroy virally infected cells and tumor cells, and arealso implicated in transplant rejection. CTLs express the CD8 moleculeat their surface.

These cells recognize their targets by binding to antigen associatedwith MHC class I, which is present on the surface of all nucleatedcells. Through IL-10, adenosine and other molecules secreted byregulatory T cells, the CD8+ cells can be inactivated to an anergicstate, which prevent autoimmune diseases such as experimental autoimmuneencephalomyelitis.

Memory T cells are a subset of antigen-specific T cells that persistlong-term after an infection has resolved. They quickly expand to largenumbers of effector T cells upon re-exposure to their cognate antigen,thus providing the immune system with “memory” against past infections.Memory T cells comprise three subtypes: central memory T cells (TCMcells) and two types of effector memory T cells (TEM cells and TEMRAcells). Memory cells may be either CD4+ or CD8+. Memory T cellstypically express the cell surface protein CD45RO. Regulatory T cells(Treg cells), formerly known as suppressor T cells, are crucial for themaintenance of immunological tolerance. Their major role is to shut downT cell-mediated immunity toward the end of an immune reaction and tosuppress auto-reactive T cells that escaped the process of negativeselection in the thymus.

Two major classes of CD4+ Treg cells have been described—naturallyoccurring Treg cells and adaptive Treg cells. Naturally occurring Tregcells (also known as CD4⁺CD25⁺FoxP3⁺ Treg cells) arise in the thymus andhave been linked to interactions between developing T cells with bothmyeloid (CD11c⁺) and plasmacytoid (CD123⁺) dendritic cells that havebeen activated with TSLP. Naturally occurring Treg cells can bedistinguished from other T cells by the presence of an intracellularmolecule called FoxP3. Adaptive Treg cells (also known as Tr1 cells orTh3 cells) may originate during a normal immune response.

Natural Killer Cells (or NK cells) are a type of cytolytic cell whichform part of the innate immune system. NK cells provide rapid responsesto innate signals from virally infected cells in an MHC independentmanner. NK cells (belonging to the group of innate lymphoid cells) aredefined as large granular lymphocytes (LGL) and constitute the thirdkind of cells differentiated from the common lymphoid progenitorgenerating B and T lymphocytes.

In some embodiments, therapeutic cells comprise autologous cellsengineered to express a CoStAR. In some embodiments, therapeutic cellscomprise allogeneic cells engineered to express a CoStAR. Autologouscells expressing CoStARs may be advantageous in avoidinggraft-versus-host disease (GVHD) due to TCR-mediated recognition ofrecipient alloantigens. Also, the immune system of a CoStAR recipientcould attack the infused CoStAR cells, causing rejection. In certainembodiments, to prevent GVHD, and to reduce rejection, endogenous TcR isremoved from allogeneic CoStAR cells by genome editing.

Nucleic Acids

In some embodiments, provided is a nucleic acid sequence encoding any ofthe CoStARs, polypeptides, or proteins described herein (includingfunctional portions and functional variants thereof). As used herein,the terms “polynucleotide”, “nucleotide”, and “nucleic acid” areintended to be synonymous with each other. It will be understood by askilled person that numerous different polynucleotides and nucleic acidscan encode the same polypeptide as a result of the degeneracy of thegenetic code. In addition, it is to be understood that skilled personsmay, using routine techniques, make nucleotide substitutions that do notaffect the polypeptide sequence encoded by the polynucleotides describedhere to reflect the codon usage of any particular host organism in whichthe polypeptides are to be expressed, e.g. codon optimisation. Nucleicacids may comprise DNA or RNA. They may be single stranded ordouble-stranded. They may also be polynucleotides which include withinthem synthetic or modified nucleotides. A number of different types ofmodification to oligonucleotides are known in the art. These includemethylphosphonate and phosphorothioate backbones, addition of acridineor polylysine chains at the 3′ and/or 5′ ends of the molecule. For thepurposes of the present disclosure, it is to be understood that thepolynucleotides may be modified by any method available in the art. Suchmodifications may be carried out in order to enhance the in vivoactivity or life span of polynucleotides of interest.

The terms “variant”, “homologue” or “derivative” in relation to anucleotide sequence include any substitution of, variation of,modification of, replacement of, deletion of or addition of one (ormore) nucleic acid from or to the sequence.

The nucleic acid sequence may encode the CoStAR and/or fusion proteinsincluding without limitation any one of SEQ ID NOS:42-247 or SEQ IDNOS:196-335, or a variant thereof. The nucleotide sequence may comprisea codon optimised nucleic acid sequence shown engineered for expressionin human cells.

Provided is a nucleic acid sequence which comprises a nucleic acidsequence encoding a CoStAR and a further nucleic acid sequence encodinga T-cell receptor (TCR) and/or chimeric antigen receptor (CAR).

The nucleic acid sequences may be joined by a sequence allowingco-expression of the two or more nucleic acid sequences. For example,the construct may comprise an internal promoter, an internal ribosomeentry sequence (IRES) sequence or a sequence encoding a cleavage site.The cleavage site may be self-cleaving, such that when the polypeptideis produced, it is immediately cleaved into the discrete proteinswithout the need for any external cleavage activity. Variousself-cleaving sites are known, including the Foot- and Mouth diseasevirus (FMDV) and the 2A self-cleaving peptide. The co-expressingsequence may be an internal ribosome entry sequence (IRES). Theco-expressing sequence may be an internal promoter.

Vectors

In some embodiments, provided is a vector which comprises a nucleic acidsequence or nucleic acid.

Such a vector may be used to introduce the nucleic acid sequence(s) ornucleic acid construct(s) into a host cell so that it expresses one ormore CoStAR(s) according to the first aspect of the invention and,optionally, one or more other proteins of interest (POI), for example aTCR or a CAR. The vector may, for example, be a plasmid or a viralvector, such as a retroviral vector or a lentiviral vector, or atransposon-based vector or synthetic mRNA.

The nucleic acids of the present invention may also be used for nucleicacid immunization and gene therapy, using standard gene deliveryprotocols. Methods for gene delivery are known in the art. See, e.g.,U.S. Pat. Nos. 5,399,346, 5,580,859, 5,589,466, incorporated byreference herein in their entireties.

Vectors derived from retroviruses, such as the lentivirus, are suitabletools to achieve long-term gene transfer since they allow long-term,stable integration of a transgene or transgenes and its propagation indaughter cells. The vector may be capable of transfecting or transducinga lymphocyte including a T cell or an NK cell. Also provided are vectorsin which a nucleic acid as provided herein is inserted. The expressionof natural or synthetic nucleic acids encoding a CoStAR, and optionallya TCR or CAR is typically achieved by operably linking a nucleic acidencoding the CoStAR and TCR/CAR polypeptide or portions thereof to oneor more promoters, and incorporating the construct into an expressionvector.

Additional promoter elements, e.g., enhancers, regulate the frequency oftranscriptional initiation. Typically, these are located in the region30-110 bp upstream of the start site, although a number of promotershave recently been shown to contain functional elements downstream ofthe start site as well. The spacing between promoter elements frequentlyis flexible, so that promoter function is preserved when elements areinverted or moved relative to one another. In the thymidine kinase (tk)promoter, the spacing between promoter elements can be increased to 50bp apart before activity begins to decline.

One example of a suitable promoter is the immediate earlycytomegalovirus (CMV) promoter sequence. This promoter sequence is astrong constitutive promoter sequence capable of driving high levels ofexpression of any polynucleotide sequence operatively linked thereto.Another example of a suitable promoter is Elongation Growth Factor-1α(EF-1α). However, other constitutive promoter sequences may also beused, including, but not limited to the simian virus 40 (SV40) earlypromoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus(HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, MSCVpromoter, MND promoter, an avian leukemia virus promoter, anEpstein-Barr virus immediate early promoter, a Rous sarcoma viruspromoter, as well as human gene promoters such as, but not limited to,the actin promoter, the myosin promoter, the hemoglobin promoter, andthe creatine kinase promoter.

The vectors can be suitable for replication and integration ineukaryotic cells. Typical cloning vectors contain transcription andtranslation terminators, initiation sequences, and promoters useful forregulation of the expression of the desired nucleic acid sequence. Viralvector technology is well known in the art and is described, forexample, in Sambrook et al. (2001, Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory, New York), and in other virologyand molecular biology manuals, see also, WO 01/96584; WO 01/29058; andU.S. Pat. No. 6,326,193). In some embodiments, the constructs expressedare as shown in SEQ ID NOS:42-247, or 196-335, 362-363, 369-420, and514. In some embodiments the nucleic acids are multi-cistronicconstructs that permit the expression of multiple transgenes (e.g.,CoStAR and a TCR and/or CAR etc.) under the control of a singlepromoter. In some embodiments, the transgenes (e.g., CoStAR and a TCRand/or CAR etc.) are separated by a self-cleaving 2A peptide. Examplesof 2A peptides useful in the nucleic acid constructs of the include F2A,P2A, T2A and E2A. In other embodiments, the nucleic acid construct is amulti-cistronic construct comprising two promoters; one promoter drivingthe expression of CoStAR and the other promoter driving the expressionof the TCR or CAR. In some embodiments, the dual promoter constructs areuni-directional. In some embodiments, the dual promoter constructs arebi-directional. In order to assess the expression of the CoStARpolypeptide or portions thereof, the expression vector to be introducedinto a cell can also contain either a selectable marker gene or areporter gene or both to facilitate identification and selection ofexpressing cells from the population of cells sought to be transfectedor transduced through viral vectors.

Sources of Cells

Prior to expansion and genetic modification, a source of cells (e.g.,immune effector cells, e.g., T cells or NK cells) is obtained from asubject. The term “subject” is intended to include living organisms inwhich an immune response can be elicited (e.g., mammals). Examples ofsubjects include humans, dogs, cats, mice, rats, and transgenic speciesthereof. T cells can be obtained from a number of sources, includingperipheral blood mononuclear cells, bone marrow, lymph node tissue, cordblood, thymus tissue, tissue from a site of infection, ascites, pleuraleffusion, spleen tissue, and tumors.

In one aspect, T cells are isolated from peripheral blood lymphocytes bylysing the red blood cells and depleting the monocytes, for example, bycentrifugation through a PERCOLL™ gradient or by counterflow centrifugalelutriation. T cell may be collected at an apheresis center and cellstorage facility where T cells can be harvested, maintained, and easilytransferred. The T cells can be cryopreserved and stored for later use.An acceptable duration of storage may be determined and validated andcan be up to 6 months, up to a year, or longer.

In another aspect, Tumor infiltrating cells (TILs) are isolated and/orexpanded from a tumor, for example by a fragmented, dissected, or enzymedigested tumor biopsy or mass. The TILs may be produced in a two-stageprocess using a tumor biopsy as the starting material: Stage 1(generally performed over 2-3 hours) initial collection and processingof tumor material using dissection, enzymatic digestion andhomogenization to produce a single cell suspension which can be directlycryopreserved to stabilize the starting material for subsequentmanufacture and Stage 2 which can occur days or years later. Stage 2 maybe performed over 4 weeks, which may be a continuous process startingwith thawing of the product of Stage 1 and growth of the TIL out of thetumor starting material (about 2 weeks) followed by a rapid expansionprocess of the TIL cells (about 2 weeks) to increase the amount of cellsand therefore dose. The TILs maybe concentrated and washed prior toformulation as a liquid suspension of cells.

The TIL population can be transduced at any point following collection.In some embodiments, a cryopreserved TIL population is transduced toexpress a CoStAR following thawing. In some embodiments, a TILpopulation is transduced to express a CoStAR during outgrowth or initialexpansion from tumor starting material. In some embodiments, a TILpopulation is transduced to express a CoStAR during REP, for example butnot limited to from about day 8 to about day 10 of REP. An exemplary TILpreparation is described in Applicant's U.S. patent application Ser. No.62/951,559, filed Dec. 20, 2019.

A specific subpopulation of T cells, such as CD3+, CD28+, CD4+, CD8+,CD45RA+, and CD45RO+T cells, can be further isolated by positive ornegative selection techniques. For example, in one aspect, T cells areisolated by incubation with anti-CD3/anti-CD28-conjugated beads, such asDYNABEADS® M-450 CD3/CD28 T, for a time period sufficient for positiveselection of the desired T cells. In one aspect, the time period isabout 30 minutes. In a further aspect, the time period ranges from 30minutes to 36 hours or longer and all integer values there between. In afurther aspect, the time period is at least 1, 2, 3, 4, 5, or 6 hours.In yet another preferred aspect, the time period is 10 to 24 hours. Inone aspect, the incubation time period is 24 hours. Longer incubationtimes may be used to isolate T cells in any situation where there arefew T cells as compared to other cell types, such in isolating tumorinfiltrating lymphocytes (TIL) from tumor tissue or fromimmunocompromised individuals. Further, use of longer incubation timescan increase the efficiency of capture of CD8+ T cells. Thus, by simplyshortening or lengthening the time T cells are allowed to bind to theCD3/CD28 beads and/or by increasing or decreasing the ratio of beads toT cells (as described further herein), subpopulations of T cells can bepreferentially selected for or against at culture initiation or at othertime points during the process. Additionally, by increasing ordecreasing the ratio of anti-CD3 and/or anti-CD28 antibodies on thebeads or other surface, subpopulations of T cells can be preferentiallyselected for or against at culture initiation or at other desired timepoints. The skilled artisan would recognize that multiple rounds ofselection can also be used in the context of this disclosure. In certainaspects, it may be desirable to perform the selection procedure and usethe “unselected” cells in the activation and expansion process.“Unselected” cells can also be subjected to further rounds of selection.

Enrichment of a T cell population by negative selection can beaccomplished with a combination of antibodies directed to surfacemarkers unique to the negatively selected cells. One method is cellsorting and/or selection via negative magnetic immunoadherence or flowcytometry that uses a cocktail of monoclonal antibodies directed to cellsurface markers present on the cells negatively selected. For example,to enrich for CD4+ cells by negative selection, a monoclonal antibodycocktail typically includes antibodies to CD14, CD20, CD16, HLA-DR, andCD8. In certain aspects, it may be desirable to enrich for or positivelyselect for regulatory T cells which typically express CD4+, CD25+,CD62Lhi, GITR+, CD137, PD1, TIM3, LAG-3, CD150 and FoxP3+.Alternatively, in certain aspects, T regulatory cells are depleted byanti-CD25 conjugated beads or other similar method of selection.

The methods described herein can include, e.g., selection of a specificsubpopulation of immune effector cells, e.g., T cells, that are a Tregulatory cell-depleted population, CD25+ depleted cells, using, e.g.,a negative selection technique, e.g., described herein. Preferably, thepopulation of T regulatory depleted cells contains less than 30%, 25%,20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% of CD25+ cells.

A specific subpopulation of CoStAR effector cells that specifically bindto a target antigen can be enriched for by positive selectiontechniques. For example, in some embodiments, effector cells areenriched for by incubation with target antigen-conjugated beads for atime period sufficient for positive selection of the desired abTCReffector cells. In some embodiments, the time period is about 30minutes. In some embodiments, the time period ranges from 30 minutes to36 hours or longer (including all ranges between these values). In someembodiments, the time period is at least one, 2, 3, 4, 5, or 6 hours. Insome embodiments, the time period is 10 to 24 hours. In someembodiments, the incubation time period is 24 hours. For isolation ofeffector cells present at low levels in the heterogeneous cellpopulation, use of longer incubation times, such as 24 hours, canincrease cell yield. Longer incubation times may be used to isolateeffector cells in any situation where there are few effector cells ascompared to other cell types. The skilled artisan would recognize thatmultiple rounds of selection can also be used in the context of thisdisclosure.

T cells for stimulation can also be frozen after a washing step. Afterthe washing step that removes plasma and platelets, the cells may besuspended in a freezing solution. While many freezing solutions andparameters are known in the art and will be useful in this context, onemethod involves using PBS containing 20% DMSO and 8% human serumalbumin, or culture media containing 10% Dextran 40 and 5% Dextrose, 20%Human Serum Albumin and 7.5% DMSO, or 31.25% Plasmalyte-A, 31.25%Dextrose 5%, 0.45% NaCl, 10% Dextran 40 and 5% Dextrose, 20% Human SerumAlbumin, and 7.5% DMSO or other suitable cell freezing media containingfor example, Hespan and PlasmaLyte A, the cells then are frozen to −80°C. at a rate of 1° per minute and stored in the vapor phase of a liquidnitrogen storage tank. Other methods of controlled freezing may be usedas well as uncontrolled freezing immediately at −20° C. or in liquidnitrogen.

Allogeneic CoStAR

In embodiments described herein, the immune effector cell can be anallogeneic immune effector cell, e.g., T cell or NK cell. For example,the cell can be an allogeneic T cell, e.g., an allogeneic T cell lackingexpression of endogenous T cell receptor (TCR) and/or human leukocyteantigen (HLA), e.g., HLA class I and/or HLA class II.

A T cell lacking a functional endogenous TCR can be, e.g., engineeredsuch that it does not express any functional TCR on its surface,engineered such that it does not express one or more subunits thatcomprise a functional TCR (e.g., engineered such that it does notexpress (or exhibits reduced expression) of TCR alpha, TCR beta, TCRgamma, TCR delta, TCR epsilon, and/or TCR zeta) or engineered such thatit produces very little functional TCR on its surface. Alternatively,the T cell can express a substantially impaired TCR, e.g., by expressionof mutated or truncated forms of one or more of the subunits of the TCR.The term “substantially impaired TCR” means that this TCR will notelicit an adverse immune reaction in a host.

A T cell described herein can be, e.g., engineered such that it does notexpress a functional HLA on its surface. For example, a T cell describedherein, can be engineered such that cell surface expression HLA, e.g.,HLA class 1 and/or HLA class II, is downregulated. In some aspects,downregulation of HLA may be accomplished by reducing or eliminatingexpression of beta-2 microglobulin (B2M).

In some embodiments, the T cell can lack a functional TCR and afunctional HLA, e.g., HLA class I and/or HLA class II. Modified T cellsthat lack expression of a functional TCR and/or HLA can be obtained byany suitable means, including a knock out or knock down of one or moresubunit of TCR or HLA. For example, the T cell can include a knock downof TCR and/or HLA using siRNA, shRNA, clustered regularly interspacedshort palindromic repeats (CRISPR) transcription-activator like effectornuclease (TALEN), or zinc finger endonuclease (ZFN).

In some embodiments, the allogeneic cell can be a cell which does notexpresses or expresses at low levels an inhibitory molecule, e.g. a cellengineered by any method described herein. For example, the cell can bea cell that does not express or expresses at low levels an inhibitorymolecule, e.g., that can decrease the ability of a CoStAR-expressingcell to mount an immune effector response. Examples of inhibitorymolecules include PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g.,CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1,CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 orCD270), KIR, A2aR, MHC class I, MHC class II, Gal9, adenosine, and TGFRbeta. Inhibition of an inhibitory molecule, e.g., by inhibition at theDNA, RNA or protein level, can optimize a CAR-expressing cellperformance. In embodiments, an inhibitory nucleic acid, e.g., aninhibitory nucleic acid, e.g., a dsRNA, e.g., an siRNA or shRNA, aclustered regularly interspaced short palindromic repeats (CRISPR), atranscription-activator like effector nuclease (TALEN), or a zinc fingerendonuclease (ZFN), e.g., as described herein, can be used.

Use of siRNA or shRNA to Inhibit Endogenous TCR or HLA

In some embodiments, TCR expression and/or HLA expression can beinhibited using siRNA or shRNA that targets a nucleic acid encoding aTCR and/or HLA, and/or an inhibitory molecule described herein (e.g.,PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/orCEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86,B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHCclass I, MHC class II, Gal9, adenosine, and TGFR beta), in a T cell.

Expression of siRNA and shRNAs in T cells can be achieved using anyconventional expression system, e.g., such as a lentiviral expressionsystem. Exemplary shRNAs that downregulate expression of components ofthe TCR are described, e.g., in US Publication No.: 2012/0321667.Exemplary siRNA and shRNA that downregulate expression of HLA class Iand/or HLA class II genes are described, e.g., in U.S. publication No.:US 2007/0036773.

Crispr to Inhibit TCR or HLA

“CRISPR” or “CRISPR to inhibit TCR and/or HLA” as used herein refers toa set of clustered regularly interspaced short palindromic repeats, or asystem comprising such a set of repeats. “Cas”, as used herein, refersto a CRISPR-associated protein. A “CRISPR/Cas” system refers to a systemderived from CRISPR and Cas which can be used to silence or mutate a TCRand/or HLA gene, and/or an inhibitory molecule described herein (e.g.,PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/orCEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86,B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHCclass I, MHC class II, GAL9, adenosine, and TGFR beta).

Naturally-occurring CRISPR/Cas systems are found in approximately 40% ofsequenced eubacteria genomes and 90% of sequenced archaea. Grissa et al.(2007) BMC Bioinformatics 8: 172. This system is a type of prokaryoticimmune system that confers resistance to foreign genetic elements suchas plasmids and phages and provides a form of acquired immunity.Barrangou et al. (2007) Science 315: 1709-1712; Marragini et al. (2008)Science 322: 1843-1845.

In some embodiments, the fusion protein or CoSTaR comprises polypeptidesof SEQ ID NO: 192, 210, 228, 246, 264, 282, and/or any one of SEQ ID NO:348 where the sequences are shown in Table 7. In some embodiments, thisincludes some part of SEQ ID NO: 192, 210, 228, 246, 264, 282 and/orparts of SEQ ID NO: 348, and/or variants thereof. In some embodiments,the fusion protein or CoSTaR comprises the CEA construct componentsprovided in FIG. 83 (all or in part or variants thereof). In someembodiments, the fusion protein or CoSTaR comprises the MSLN constructcomponents provided in FIG. 84 (all or in part or variants thereof). Insome embodiments, this includes SEQ ID Nos: 83 (all or in part orvariants thereof). In some embodiments, this includes SEQ ID Nos: 84(all or in part or variants thereof). In some embodiments, the fusionprotein or CoStAR will be the same as shown in Table 7, with the bindingdomain (such as an scFv) to MSLN or CEA.

In some embodiments, a cancer specific CAR or TCR is present in the cellthat contains the fusion protein or CoStAR. In some embodiments, afusion protein or CoStAR can be expressed alone under the control of apromoter in a therapeutic population of cells that have therapeuticactivity, for example, Tumor Infiltrating Lymphocytes (TILs). In someembodiments, the fusion protein or CoStAR can be expressed along with atherapeutic transgene such as a chimeric antigen receptor (CAR) and/orT-cell Receptor (TCR).

In some embodiments, suitable TCRs and CARs can be those that are wellknown in the literature, for example HLA-A*02-NYESO-1 specific TCRs(Rapoport et al. Nat Med 2015) or anti-CD19scFv.CD3z fusion CARs(Kochenderfer et al. J Clin Oncol 2015) which have been successfullyused to treat Myeloma or B-cell malignancies respectively. In someembodiments, the CoStARs described herein can be expressed with anyknown CAR or TCR thus providing the cell with a regulatable growthswitch to allow cell expansion in-vitro or in-vivo, and a conventionalactivation mechanism in the form of the TCR or CAR for anti-canceractivity. In some embodiments, a cell for use in adoptive cell therapyis provided and comprises a CoStAR as described herein and a TCR and/orCAR that specifically binds to a tumor associated antigen. In someembodiments, an exemplary CoStAR comprising CD28 includes anextracellular antigen binding domain and an extracellular, transmembraneand intracellular signaling domain.

Activation and Expansion of T Cells

T cells may be activated and expanded generally using methods asdescribed, for example, in U.S. Pat. Nos. 6,352,694; 6,534,055;6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575;7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874;6,797,514; 6,867,041; and U.S. Patent Application Publication No.20060121005.

Generally, the T cells may be expanded by contact with a surface havingattached thereto an agent that stimulates a CD3/TCR complex associatedsignal and a ligand that stimulates a costimulatory molecule on thesurface of the T cells. In particular, T cell populations may bestimulated as described herein, such as by contact with an anti-CD3antibody, or antigen-binding fragment thereof, or an anti-CD2 antibodyimmobilized on a surface, or by contact with a protein kinase Cactivator (e.g., bryostatin) in conjunction with a calcium ionophore.For co-stimulation of an accessory molecule on the surface of the Tcells, a ligand that binds the accessory molecule is used. For example,a population of T cells can be contacted with an anti-CD3 antibody andan anti-CD28 antibody, under conditions appropriate for stimulatingproliferation of the T cells. To stimulate proliferation of either CD4+T cells or CD8+ T cells, an anti-CD3 antibody and an anti-CD28 antibodycan be used. Examples of an anti-CD28 antibody include 9.3, B-T3,XR-CD28 (Diaclone, Besancon, France) can be used as can other methodscommonly known in the art (Berg et al., Transplant Proc.30(8):3975-3977, 1998; Haanen et al., J. Exp. Med. 190(9):13191328,1999; Garland et al., J. Immunol Meth. 227(1-2):53-63, 1999).

In some embodiments, expansion can be performed using flasks orcontainers, or gas-permeable containers known by those of skill in theart and can proceed for 7 days, 8 days, 9 days, 10 days, 11 days, 12days, 13 days, or 14 days, about 7 days to about 14 days, about 8 daysto about 14 days, about 9 days to about 14 days, about 10 days to about14 days, about 11 days to about 14 days, about 12 days to about 14 days,or about 13 days to about 14 days. In some embodiments, the second TILexpansion can proceed for about 14 days.

In some embodiments, the expansion can be performed using non-specificT-cell receptor stimulation in the presence of interleukin-2 (IL-2) orinterleukin-15 (IL-15). The non-specific T-cell receptor stimulus caninclude, for example, an anti-CD3 antibody, such as about 30 ng/ml ofOKT3, a mouse monoclonal anti-CD3 antibody (commercially available fromOrtho-McNeil, Raritan, N.J. or Miltenyi Biotech, Auburn, Calif.) orUHCT-1 (commercially available from BioLegend, San Diego, Calif., USA).CoStAR cells can be expanded in vitro by including one or more antigens,including antigenic portions thereof, such as epitope(s), of a cancer,which can be optionally expressed from a vector, such as a humanleukocyte antigen A2 (HLA-A2) binding peptide, e.g., 0.3 .mu.MMART-1:26-35 (27 L) or gp100:209-217 (210M), optionally in the presenceof a T-cell growth factor, such as 300 IU/mL IL-2 or IL-15. Othersuitable antigens may include, e.g., NY-ESO-1, TRP-1, TRP-2, tyrosinasecancer antigen, MAGE-A3, SSX-2, and VEGFR2, or antigenic portionsthereof. CoStAR cells may also be rapidly expanded by restimulation withthe same antigen(s) of the cancer pulsed onto HLA-A2-expressingantigen-presenting cells. Alternatively, the CoStAR cells can be furtherstimulated with, e.g., example, irradiated, autologous lymphocytes orwith irradiated HLA-A2+ allogeneic lymphocytes and IL-2. In someembodiments, the stimulation occurs as part of the expansion. In someembodiments, the expansion occurs in the presence of irradiated,autologous lymphocytes or with irradiated HLA-A2+ allogeneic lymphocytesand IL-2.

In some embodiments, the cell culture medium comprises IL-2. In someembodiments, the cell culture medium comprises about 1000 IU/mL, about1500 IU/mL, about 2000 IU/mL, about 2500 IU/mL, about 3000 IU/mL, about3500 IU/mL, about 4000 IU/mL, about 4500 IU/mL, about 5000 IU/mL, about5500 IU/mL, about 6000 IU/mL, about 6500 IU/mL, about 7000 IU/mL, about7500 IU/mL, or about 8000 IU/mL, or between 1000 and 2000 IU/mL, between2000 and 3000 IU/mL, between 3000 and 4000 IU/mL, between 4000 and 5000IU/mL, between 5000 and 6000 IU/mL, between 6000 and 7000 IU/mL, between7000 and 8000 IU/mL, or between 8000 IU/mL of IL-2.

In some embodiments, the cell culture medium comprises OKT3 antibody. Insome embodiments, the cell culture medium comprises about 0.1 ng/mL,about 0.5 ng/mL, about 1 ng/mL, about 2.5 ng/mL, about 5 ng/mL, about7.5 ng/mL, about 10 ng/mL, about 15 ng/mL, about 20 ng/mL, about 25ng/mL, about 30 ng/mL, about 35 ng/mL, about 40 ng/mL, about 50 ng/mL,about 60 ng/mL, about 70 ng/mL, about 80 ng/mL, about 90 ng/mL, about100 ng/mL, about 200 ng/mL, about 500 ng/mL, about 1 μg/mL or between0.1 ng/mL and 1 ng/mL, between 1 ng/mL and 5 ng/mL, between 5 ng/mL and10 ng/mL, between 10 ng/mL and 20 ng/mL, between 20 ng/mL and 30 ng/mL,between 30 ng/mL and 40 ng/mL, between 40 ng/mL and 50 ng/mL, or between50 ng/mL and 100 ng/mL of OKT3 antibody.

In some embodiments, a combination of IL-2, IL-7, IL-15, and/or IL-21are employed as a combination during the expansion. In some embodiments,IL-2, IL-7, IL-15, and/or IL-21 as well as any combinations thereof canbe included during the expansion. In some embodiments, a combination ofIL-2, IL-15, and IL-21 are employed as a combination during theexpansion. In some embodiments, IL-2, IL-15, and IL-21 as well as anycombinations thereof can be included.

In some embodiments, the expansion can be conducted in a supplementedcell culture medium comprising IL-2, OKT-3, and antigen-presentingfeeder cells.

In some embodiments, the expansion culture media comprises about 500IU/mL of IL-15, about 400 IU/mL of IL-15, about 300 IU/mL of IL-15,about 200 IU/mL of IL-15, about 180 IU/mL of IL-15, about 160 IU/mL ofIL-15, about 140 IU/mL of IL-15, about 120 IU/mL of IL-15, or about 100IU/mL of IL-15, or about 500 IU/mL of IL-15 to about 100 IU/mL of IL-15,or about 400 IU/mL of IL-15 to about 100 IU/mL of IL-15 or about 300IU/mL of IL-15 to about 100 IU/mL of IL-15 or about 200 IU/mL of IL-15,or about 180 IU/mL of IL-15.

In some embodiments, the expansion culture media comprises about 20IU/mL of IL-21, about 15 IU/mL of IL-21, about 12 IU/mL of IL-21, about10 IU/mL of IL-21, about 5 IU/mL of IL-21, about 4 IU/mL of IL-21, about3 IU/mL of IL-21, about 2 IU/mL of IL-21, about 1 IU/mL of IL-21, orabout 0.5 IU/mL of IL-21, or about 20 IU/mL of IL-21 to about 0.5 IU/mLof IL-21, or about 15 IU/mL of IL-21 to about 0.5 IU/mL of IL-21, orabout 12 IU/mL of IL-21 to about 0.5 IU/mL of IL-21, or about 10 IU/mLof IL-21 to about 0.5 IU/mL of IL-21, or about 5 IU/mL of IL-21 to about1 IU/mL of IL-21, or about 2 IU/mL of IL-21. In some embodiments, thecell culture medium comprises about 1 IU/mL of IL-21, or about 0.5 IU/mLof IL-21.

In some embodiments the antigen-presenting feeder cells (APCs) arePBMCs. In an embodiment, the ratio of CoStAR cells to PBMCs and/orantigen-presenting cells in the expansion is about 1 to 25, about 1 to50, about 1 to 100, about 1 to 125, about 1 to 150, about 1 to 175,about 1 to 200, about 1 to 225, about 1 to 250, about 1 to 275, about 1to 300, about 1 to 325, about 1 to 350, about 1 to 375, about 1 to 400,or about 1 to 500, or between 1 to 50 and 1 to 300, or between 1 to 100and 1 to 200.

In certain aspects, the primary stimulatory signal and the costimulatorysignal for the T cell may be provided by different protocols. Forexample, the agents providing each signal may be in solution or coupledto a surface. When coupled to a surface, the agents may be coupled tothe same surface (i.e., in “cis” formation) or to separate surfaces(i.e., in “trans” formation). Alternatively, one agent may be coupled toa surface and the other agent in solution. In one aspect, the agentproviding the costimulatory signal is bound to a cell surface and theagent providing the primary activation signal is in solution or coupledto a surface. In certain aspects, both agents can be in solution. In oneaspect, the agents may be in soluble form, and then cross-linked to asurface, such as a cell expressing Fc receptors or an antibody or otherbinding agent which will bind to the agents. In this regard, see forexample, U.S. Patent Application Publication Nos. 20040101519 and20060034810 for artificial antigen presenting cells (aAPCs) that arecontemplated for use in activating and expanding T cells in the presentdisclosure.

In one aspect, the two agents are immobilized on beads, either on thesame bead, i.e., “cis,” or to separate beads, i.e., “trans.” By way ofexample, the agent providing the primary activation signal is ananti-CD3 antibody or an antigen-binding fragment thereof and the agentproviding the costimulatory signal is an anti-CD28 antibody orantigen-binding fragment thereof; and both agents are co-immobilized tothe same bead in equivalent molecular amounts. In one aspect, a 1:1ratio of each antibody bound to the beads for CD4+ T cell expansion andT cell growth is used. In some embodiments, a ratio of anti CD3:CD28antibodies bound to the beads is used such that an increase in T cellexpansion is observed as compared to the expansion observed using aratio of 1:1. In one particular aspect an increase of from about 1 toabout 3 fold is observed as compared to the expansion observed using aratio of 1:1. In one aspect, the ratio of CD3:CD28 antibody bound to thebeads ranges from 100:1 to 1:100 and all integer values there between.In some embodiments, more anti-CD28 antibody is bound to the particlesthan anti-CD3 antibody, i.e., the ratio of CD3:CD28 is less than one. Insome embodiments, the ratio of anti CD28 antibody to anti CD3 antibodybound to the beads is greater than 2:1. In one particular aspect, a1:100 CD3:CD28 ratio of antibody bound to beads is used. In one aspect,a 1:75 CD3:CD28 ratio of antibody bound to beads is used. In a furtheraspect, a 1:50 CD3:CD28 ratio of antibody bound to beads is used. In oneaspect, a 1:30 CD3:CD28 ratio of antibody bound to beads is used. In onepreferred aspect, a 1:10 CD3:CD28 ratio of antibody bound to beads isused. In one aspect, a 1:3 CD3:CD28 ratio of antibody bound to the beadsis used. In yet one aspect, a 3:1 CD3:CD28 ratio of antibody bound tothe beads is used.

Ratios of particles to cells from 1:500 to 500:1 and any integer valuesin between may be used to stimulate T cells or other target cells. Asthose of ordinary skill in the art can readily appreciate, the ratio ofparticles to cells may depend on particle size relative to the targetcell. For example, small sized beads could only bind a few cells, whilelarger beads could bind many. In certain aspects the ratio of cells toparticles ranges from 1:100 to 100:1 and any integer values in-betweenand in further aspects the ratio comprises 1:9 to 9:1 and any integervalues in between, can also be used to stimulate T cells. The ratio ofanti-CD3- and anti-CD28-coupled particles to T cells that result in Tcell stimulation can vary as noted above, however certain preferredvalues include 1:100, 1:50, 1:40, 1:30, 1:20, 1:10, 1:9, 1:8, 1:7, 1:6,1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1,and 15:1 with one preferred ratio being at least 1:1 particles per Tcell. In one aspect, a ratio of particles to cells of 1:1 or less isused. In one particular aspect, a preferred particle:cell ratio is 1:5.In further aspects, the ratio of particles to cells can be varieddepending on the day of stimulation. For example, in one aspect, theratio of particles to cells is from 1:1 to 10:1 on the first day andadditional particles are added to the cells every day or every other daythereafter for up to 10 days, at final ratios of from 1:1 to 1:10 (basedon cell counts on the day of addition). In one particular aspect, theratio of particles to cells is 1:1 on the first day of stimulation andadjusted to 1:5 on the third and fifth days of stimulation. In oneaspect, particles are added on a daily or every other day basis to afinal ratio of 1:1 on the first day, and 1:5 on the third and fifth daysof stimulation. In one aspect, the ratio of particles to cells is 2:1 onthe first day of stimulation and adjusted to 1:10 on the third and fifthdays of stimulation. In one aspect, particles are added on a daily orevery other day basis to a final ratio of 1:1 on the first day, and 1:10on the third and fifth days of stimulation. One of skill in the art willappreciate that a variety of other ratios may be suitable for use. Inparticular, ratios will vary depending on particle size and on cell sizeand type. In one aspect, the most typical ratios for use are in theneighborhood of 1:1, 2:1 and 3:1 on the first day.

In further aspects, the cells, such as T cells, are combined withagent-coated beads, the beads and the cells are subsequently separated,and then the cells are cultured. In an alternative aspect, prior toculture, the agent-coated beads and cells are not separated but arecultured together. In a further aspect, the beads and cells are firstconcentrated by application of a force, such as a magnetic force,resulting in increased ligation of cell surface markers, therebyinducing cell stimulation.

Preparation of CoStAR Cells

Viral- and non-viral-based genetic engineering tools can be used togenerate CoStAR cells, including without limitation T cells, NK cellsresulting in permanent or transient expression of therapeutic genes.Retrovirus-based gene delivery is a mature, well-characterizedtechnology, which has been used to permanently integrate CARs into thehost cell genome (Scholler J., e.g. Decade-long safety and function ofretroviral-modified chimeric antigen receptor T cells. Sci. Transl. Med.2012; 4:132ra53; Rosenberg S. A. et al., Gene transfer intohumans-immunotherapy of patients with advanced melanoma, usingtumor-infiltrating lymphocytes modified by retroviral gene transduction.N. Engl. J. Med. 1990; 323:570-578)

Non-viral DNA transfection methods can also be used. For example, Singhet al describes use of the Sleeping Beauty (SB) transposon systemdeveloped to engineer CAR T cells (Singh H., et al., Redirectingspecificity of T-cell populations for CD19 using the Sleeping Beautysystem. Cancer Res. 2008; 68:2961-2971) and is being used in clinicaltrials (see e.g., ClinicalTrials.gov: NCT00968760 and NCT01653717). Thesame technology is applicable to engineer CoStARs cells.

Multiple SB enzymes have been used to deliver transgenes. Mit6sdescribes a hyperactive transposase (SB100X) with approximately 100-foldenhancement in efficiency when compared to the first-generationtransposase. SB100X supported 35-50% stable gene transfer in humanCD34(+) cells enriched in hematopoietic stem or progenitor cells. (Mit6sL. et al., Molecular evolution of a novel hyperactive Sleeping Beautytransposase enables robust stable gene transfer in vertebrates. Nat.Genet. 2009; 41:753-761) and multiple transgenes can be delivered frommulticistronic single plasmids (e.g., Thokala R. et al., Redirectingspecificity of T cells using the Sleeping Beauty system to expresschimeric antigen receptors by mix-and-matching of VL and VH domainstargeting CD123+ tumors. PLoS ONE. 2016; 11:e0159477) or multipleplasmids (e.g., Hurton L. V. et al., Tethered IL-15 augments antitumoractivity and promotes a stem-cell memory subset in tumor-specific Tcells. Proc. Natl. Acad. Sci. USA. 2016; 113:E7788-E7797). Such systemsare used with CoStARs.

Morita et al, describes the piggyBac transposon system to integratelarger transgenes (Morita D. et al., Enhanced expression of anti-CD19chimeric antigen receptor in piggyBac transposon-engineered T cells.Mol. Ther. Methods Clin. Dev. 2017; 8:131-140) Nakazawa et al. describesuse of the system to generate EBV-specific cytotoxic T-cells expressingHER2-specific chimeric antigen receptor (Nakazawa Y et al,PiggyBac-mediated cancer immunotherapy using EBV-specific cytotoxicT-cells expressing HER2-specific chimeric antigen receptor. Mol. Ther.2011; 19:2133-2143). Manuri et al used the system to generate CD-19specific T cells (Manuri P. V. R. et al., piggyBactransposon/transposase system to generate CD19-specific T cells for thetreatment of B-lineage malignancies. Hum. Gene Ther. 2010; 21:427-437).

Transposon technology is easy and economical. One potential drawback isthe longer expansion protocols currently employed may result in T celldifferentiation, impaired activity and poor persistence of the infusedcells. Monjezi et al describe development minicircle vectors thatminimize these difficulties through higher efficiency integrations(Monjezi R. et al., Enhanced CAR T-cell engineering using non-viralSleeping Beauty transposition from minicircle vectors. Leukemia. 2017;31:186-194). These transposon technologies can be used for CoStARs.

Pharmaceutical Compositions

The present disclosure also provides a pharmaceutical compositioncontaining a vector or a CoStAR expressing cell together with apharmaceutically acceptable carrier, diluent or excipient, andoptionally one or more further pharmaceutically active polypeptidesand/or compounds.

In some embodiments, a pharmaceutical composition is provided comprisinga CoStAR described above and a pharmaceutically acceptable carrier. Insome embodiments, a pharmaceutical composition is provided comprising anucleic acid encoding a CoStAR according to any of the embodimentsdescribed above and a pharmaceutically acceptable carrier. In someembodiments, a pharmaceutical composition is provided comprising aneffector cell expressing a CoStAR described above and a pharmaceuticallyacceptable carrier. Such a formulation may, for example, be in a formsuitable for intravenous infusion.

As used herein, by “pharmaceutically acceptable” or “pharmacologicallycompatible” is meant a material that is not biologically or otherwiseundesirable, e.g., the material may be incorporated into apharmaceutical composition administered to a patient without causing anysignificant undesirable biological effects or interacting in adeleterious manner with any of the other components of the compositionin which it is contained. Pharmaceutically acceptable carriers orexcipients have preferably met the required standards of toxicologicaland manufacturing testing and/or are included on the Inactive IngredientGuide prepared by the U.S. Food and Drug administration.

In some embodiments, a population of modified T cells expressing arecombinant CoStAR is provided. A suitable population may be produced bya method described above.

The population of modified T cells may be for use as a medicament. Forexample, a population of modified T cells as described herein may beused in cancer immunotherapy therapy, for example adoptive T celltherapy.

Some embodiments provide the use of a population of modified T cells asdescribed herein for the manufacture of a medicament for the treatmentof cancer, a population of modified T cells as described herein for thetreatment of cancer, and a method of treatment of cancer may compriseadministering a population of modified T cells as described herein to anindividual in need thereof.

The population of modified T cells may be autologous i.e. the modified Tcells were originally obtained from the same individual to whom they aresubsequently administered (i.e. the donor and recipient individual arethe same). A suitable population of modified T cells for administrationto the individual may be produced by a method comprising providing aninitial population of T cells obtained from the individual, modifyingthe T cells to express a cAMP PDE or fragment thereof and an antigenreceptor which binds specifically to cancer cells in the individual, andculturing the modified T cells.

The population of modified T cells may be allogeneic i.e. the modified Tcells were originally obtained from a different individual to theindividual to whom they are subsequently administered (i.e. the donorand recipient individual are different). The donor and recipientindividuals may be HLA matched to avoid GVHD and other undesirableimmune effects. A suitable population of modified T cells foradministration to a recipient individual may be produced by a methodcomprising providing an initial population of T cells obtained from adonor individual, modifying the T cells to express a CoStAR which bindsspecifically to cancer cells in the recipient individual, and culturingthe modified T cells.

Following administration of the modified T cells, the recipientindividual may exhibit a T cell mediated immune response against cancercells in the recipient individual. This may have a beneficial effect onthe cancer condition in the individual.

Cancer conditions may be characterised by the abnormal proliferation ofmalignant cancer cells and may include leukaemias, such as AML, CML, ALLand CLL, lymphomas, such as Hodgkin lymphoma, non-Hodgkin lymphoma andmultiple myeloma, and solid cancers such as sarcomas, skin cancer,melanoma, bladder cancer, brain cancer, breast cancer, uterus cancer,ovary cancer, prostate cancer, lung cancer, colorectal cancer, cervicalcancer, liver cancer, head and neck cancer, oesophageal cancer, pancreascancer, renal cancer, adrenal cancer, stomach cancer, testicular cancer,cancer of the gall bladder and biliary tracts, thyroid cancer, thymuscancer, cancer of bone, and cerebral cancer, as well as cancer ofunknown primary (CUP).

Cancer cells within an individual may be immunologically distinct fromnormal somatic cells in the individual (i.e. the cancerous tumor may beimmunogenic). For example, the cancer cells may be capable of elicitinga systemic immune response in the individual against one or moreantigens expressed by the cancer cells. The tumor antigens that elicitthe immune response may be specific to cancer cells or may be shared byone or more normal cells in the individual.

An individual suitable for treatment as described above may be a mammal,such as a rodent (e.g. a guinea pig, a hamster, a rat, a mouse), murine(e.g. a mouse), canine (e.g. a dog), feline (e.g. a cat), equine (e.g. ahorse), a primate, simian (e.g. a monkey or ape), a monkey (e.g.marmoset, baboon), an ape (e.g. gorilla, chimpanzee, orang-utan,gibbon), or a human.

In preferred embodiments, the individual is a human. In other preferredembodiments, non-human mammals, especially mammals that areconventionally used as models for demonstrating therapeutic efficacy inhumans (e.g. murine, primate, porcine, canine, or rabbit animals) may beemployed.

Method of Treatment

Cells, including T and NK cells, expressing CoStARs can either becreated ex vivo either from a patient's own peripheral blood(autologous), or in the setting of a haematopoietic stem cell transplantfrom donor peripheral blood (allogenic), or peripheral blood from anunconnected donor (allogenic). In some embodiments, T-cells or NK cellscan be derived from ex-vivo differentiation of inducible progenitorcells or embryonic progenitor cells to T-cells or NK cells. In theseinstances, T-cells expressing a CoStAR and, optionally, a CAR and/orTCR, are generated by introducing DNA or RNA coding for the CoStAR and,optionally, a CAR and/or TCR, by one of many means includingtransduction with a viral vector, transfection with DNA or RNA.

T or NK cells expressing a CoStAR and, optionally, expressing a TCRand/or CAR can be used for the treatment of haematological cancers orsolid tumors.

In some embodiments, a method of cell therapy comprising identifying asubject in need of tumor infiltrating lymphocyte (“TIL”) cell therapyand administering to the subject a TIL cell therapy is provided. The TILcell therapy comprises a fusion protein as provided herein (such as aCEA CoStAR and/or fusion protein or a MSLN CoStAR and/or fusionprotein), which can comprise a binding domain specific (e.g, specificfor CEA CoStAR and/or fusion protein or a MSLN CoStAR and/or fusionprotein) linked to a transmembrane domain that is linked to a CD28signaling domain (for MSLN, or an ICOS domain for CEA) that is linked toa CD40 signaling domain.

The term “therapeutically effective amount” refers to an amount of aCoStAR or composition comprising a CoStAR as disclosed herein, effectiveto “treat” a disease or disorder in an individual. In the case ofcancer, the therapeutically effective amount of a CoStAR or compositioncomprising a CoStAR as disclosed herein can reduce the number of cancercells; reduce the tumor size or weight; inhibit (i.e., slow to someextent and preferably stop) cancer cell infiltration into peripheralorgans; inhibit (i.e., slow to some extent and preferably stop) tumormetastasis; inhibit, to some extent, tumor growth; and/or relieve tosome extent one or more of the symptoms associated with the cancer. Tothe extent a CoStAR or composition comprising a CoStAR as disclosedherein can prevent growth and/or kill existing cancer cells, it can becytostatic and/or cytotoxic. In some embodiments, the therapeuticallyeffective amount is a growth inhibitory amount. In some embodiments, thetherapeutically effective amount is an amount that improves progressionfree survival of a patient. In the case of infectious disease, such asviral infection, the therapeutically effective amount of a CoStAR orcomposition comprising a CoStAR as disclosed herein can reduce thenumber of cells infected by the pathogen; reduce the production orrelease of pathogen-derived antigens; inhibit (i.e., slow to some extentand preferably stop) spread of the pathogen to uninfected cells; and/orrelieve to some extent one or more symptoms associated with theinfection. In some embodiments, the therapeutically effective amount isan amount that extends the survival of a patient.

Cells, including T and NK cells, expressing CoStARs for use in themethods of the present may either be created ex vivo either from apatient's own peripheral blood (autologous), or in the setting of ahaematopoietic stem cell transplant from donor peripheral blood(allogenic), or peripheral blood from an unconnected donor (allogenic).Alternatively, T-cells or NK cells may be derived from ex-vivodifferentiation of inducible progenitor cells or embryonic progenitorcells to T-cells or NK cells. In these instances, T-cells expressing aCoStAR and, optionally, a CAR and/or TCR, are generated by introducingDNA or RNA coding for the CoStAR and, optionally, a CAR and/or TCR, byone of many means including transduction with a viral vector,transfection with DNA or RNA.

T or NK cells expressing a CoStAR as disclosed herein and, optionally,expressing a TCR and/or CAR may be used for the treatment ofhaematological cancers or solid tumors.

A method for the treatment of disease relates to the therapeutic use ofa vector or cell, including a T or NK cell, as disclosed herein. In thisrespect, the vector, or T or NK cell may be administered to a subjecthaving an existing disease or condition in order to lessen, reduce orimprove at least one symptom associated with the disease and/or to slowdown, reduce or block the progression of the disease. In someembodiments, the method can cause or promote T-cell mediated killing ofcancer cells. In some embodiments, the vector, or T or NK cell can beadministered to a patient with one or more additional therapeuticagents. The one or more additional therapeutic agents can beco-administered to the patient. By “co-administering” is meantadministering one or more additional therapeutic agents and the vector,or T or NK cell as provided herein sufficiently close in time such thatthe vector, or T or NK cell can enhance the effect of one or moreadditional therapeutic agents, or vice versa. In this regard, thevectors or cells can be administered first and the one or moreadditional therapeutic agents can be administered second, or vice versa.Alternatively, the vectors or cells and the one or more additionaltherapeutic agents can be administered simultaneously. Oneco-administered therapeutic agent that may be useful is IL-2, as this iscurrently used in existing cell therapies to boost the activity ofadministered cells. However, IL-2 treatment is associated with toxicityand tolerability issues.

As mentioned, for administration to a patient, the CoStAR effector cellscan be allogeneic or autologous to the patient. In some embodiments,allogeneic cells are further genetically modified, for example by geneediting, so as to minimize or prevent GVHD and/or a patient's immuneresponse against the CoStAR cells.

The CoStAR effector cells are used to treat cancers and neoplasticdiseases associated with a target antigen. Cancers and neoplasticdiseases that may be treated using any of the methods described hereininclude tumors that are not vascularized, or not yet substantiallyvascularized, as well as vascularized tumors. The cancers may comprisenon-solid tumors (such as hematological tumors, for example, leukemiasand lymphomas) or may comprise solid tumors. Types of cancers to betreated with the CoStAR effector cells of the include, but are notlimited to, carcinoma, blastoma, and sarcoma, and certain leukemia orlymphoid malignancies, benign and malignant tumors, and malignanciese.g., sarcomas, carcinomas, and melanomas. Adult tumors/cancers andpediatric tumors/cancers are also included.

Hematologic cancers are cancers of the blood or bone marrow. Examples ofhematological (or hematogenous) cancers include leukemias, includingacute leukemias (such as acute lymphocytic leukemia, acute myelocyticleukemia, acute myelogenous leukemia and myeloblastic, promyelocytic,myelomonocytic, monocytic and erythroleukemia), chronic leukemias (suchas chronic myelocytic (granulocytic) leukemia, chronic myelogenousleukemia, and chronic lymphocytic leukemia), polycythemia vera,lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and highgrade forms), multiple myeloma, plasmacytoma, Waldenstrom'smacroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairycell leukemia and myelodysplasia.

Solid tumors are abnormal masses of tissue that usually do not containcysts or liquid areas. Solid tumors can be benign or malignant.Different types of solid tumors are named for the type of cells thatform them (such as sarcomas, carcinomas, and lymphomas). Examples ofsolid tumors, such as sarcomas and carcinomas, include adrenocorticalcarcinoma, cholangiocarcinoma, fibrosarcoma, myxosarcoma, liposarcoma,chondrosarcoma, osteosarcoma, and other sarcomas, synovioma,mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, coloncarcinoma, stomach cancer, lymphoid malignancy, pancreatic cancer,breast cancer, lung cancers, ovarian cancer, prostate cancer,hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma,adenocarcinoma, sweat gland carcinoma, thyroid cancer (e.g., medullarythyroid carcinoma and papillary thyroid carcinoma), pheochromocytomassebaceous gland carcinoma, papillary carcinoma, papillaryadenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cellcarcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor,cervical cancer (e.g., cervical carcinoma and pre-invasive cervicaldysplasia), colorectal cancer, cancer of the anus, anal canal, oranorectum, vaginal cancer, cancer of the vulva (e.g., squamous cellcarcinoma, intraepithelial carcinoma, adenocarcinoma, and fibrosarcoma),penile cancer, oropharyngeal cancer, esophageal cancer, head cancers(e.g., squamous cell carcinoma), neck cancers (e.g., squamous cellcarcinoma), testicular cancer (e.g., seminoma, teratoma, embryonalcarcinoma, teratocarcinoma, choriocarcinoma, sarcoma, Leydig cell tumor,fibroma, fibroadenoma, adenomatoid tumors, and lipoma), bladdercarcinoma, kidney cancer, melanoma, cancer of the uterus (e.g.,endometrial carcinoma), urothelial cancers (e.g., squamous cellcarcinoma, transitional cell carcinoma, adenocarcinoma, ureter cancer,and urinary bladder cancer), and CNS tumors (such as a glioma (such asbrainstem glioma and mixed gliomas), glioblastoma (also known asglioblastoma multiforme) astrocytoma, CNS lymphoma, germinoma,medulloblastoma, Schwannoma craniopharyogioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma,neuroblastoma, retinoblastoma and brain metastases).

When “an immunologically effective amount,” “an anti-tumor effectiveamount,” “a tumor-inhibiting effective amount,” or “therapeutic amount”is indicated, the precise amount of the compositions to be administeredcan be determined by a physician with consideration of individualdifferences in age, weight, tumor size, extent of infection ormetastasis, and condition of the patient (subject). It can generally bestated that a pharmaceutical composition comprising the T cellsdescribed herein may be administered at a dosage of 10⁴ to 10⁹ cells/kgbody weight, in some instances 10⁵ to 10⁶ cells/kg body weight,including all integer values within those ranges. T cell compositionsmay also be administered multiple times at these dosages. The cells canbe administered by using infusion techniques that are commonly known inimmunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med.319:1676, 198

In some embodiments, additional aspects of the method are shown in partor whole in FIGS. 83 and 83 .

In some embodiments, the dosage can be 1×10{circumflex over ( )}9CoStAR-positive (CoStAR+) viable T cells (±20% target dose). In someembodiments, the dosage can be, or be increased to 5×10{circumflex over( )}8 CoStAR+ viable T cells (±20% target dose). In some embodiments,the dosage can be, or be increased to 3×10{circumflex over ( )}9 CoStAR+viable T cells (±20% target dose). In some embodiments, the dosage canbe, or be increased to, 6×10{circumflex over ( )}9 CoStAR+ viable Tcells (±20% target dose). In some embodiments, the dosage is at leastany one of the preceding values. In some embodiments, the dosage isbetween any two of the preceding values.

Combination Therapies

A CoStAR-expressing cell described herein may be used in combinationwith other known agents and therapies. Administered “in combination”, asused herein, means that two (or more) different treatments are deliveredto the subject during the course of the subject's affliction with thedisorder, e.g., the two or more treatments are delivered after thesubject has been diagnosed with the disorder and before the disorder hasbeen cured or eliminated or treatment has ceased for other reasons. Insome embodiments, the delivery of one treatment is still occurring whenthe delivery of the second begins, so that there is overlap in terms ofadministration. This is sometimes referred to herein as “simultaneous”or “concurrent delivery”. In other embodiments, the delivery of onetreatment ends before the delivery of the other treatment begins. Insome embodiments of either case, the treatment is more effective becauseof combined administration. For example, the second treatment is moreeffective, e.g., an equivalent effect is seen with less of the secondtreatment, or the second treatment reduces symptoms to a greater extent,than would be seen if the second treatment were administered in theabsence of the first treatment, or the analogous situation is seen withthe first treatment. In some embodiments, delivery is such that thereduction in a symptom, or other parameter related to the disorder isgreater than what would be observed with one treatment delivered in theabsence of the other. The effect of the two treatments can be partiallyadditive, wholly additive, or greater than additive. The delivery can besuch that an effect of the first treatment delivered is still detectablewhen the second is delivered.

A CoStAR-expressing cell described herein and the at least oneadditional therapeutic agent can be administered simultaneously, in thesame or in separate compositions, or sequentially. For sequentialadministration, the CAR-expressing cell described herein can beadministered first, and the additional agent can be administered second,or the order of administration can be reversed.

The CoStAR therapy and/or other therapeutic agents, procedures ormodalities can be administered during periods of active disorder, orduring a period of remission or less active disease. The CoStAR therapycan be administered before the other treatment, concurrently with thetreatment, post-treatment, or during remission of the disorder.

When administered in combination, the therapy and the additional agent(e.g., second or third agent), or all, can be administered in an amountor dose that is higher, lower or the same than the amount or dosage ofeach agent used individually, e.g., as a monotherapy. In someembodiments, the administered amount or dosage of the CoStAR therapy,the additional agent (e.g., second or third agent), or all, is lower(e.g., at least 20%, at least 30%, at least 40%, or at least 50%) thanthe amount or dosage of each agent used individually, e.g., as amonotherapy. In other embodiments, the amount or dosage of the CoStARtherapy, the additional agent (e.g., second or third agent), or all,that results in a desired effect (e.g., treatment of cancer) is lower(e.g., at least 20%, at least 30%, at least 40%, or at least 50% lower)than the amount or dosage of each agent used individually, e.g., as amonotherapy, required to achieve the same therapeutic effect.

In further aspects, a CoStAR-expressing cell described herein may beused in a treatment regimen in combination with surgery, chemotherapy,radiation, immunosuppressive agents, such as cyclosporin, azathioprine,methotrexate, mycophenolate, and FK506, antibodies, or otherimmunoablative agents such as CAMPATH, anti-CD3 antibodies or otherantibody therapies, cytoxin, fludarabine, cyclosporin, FK506, rapamycin,mycophenolic acid, steroids, FR901228, cytokines, and irradiation,peptide vaccine, such as that described in Izumoto et al. 2008 JNeurosurg 108:963-971.

In some embodiments, compounds as disclosed herein are combined withother therapeutic agents, such as other anti-cancer agents,anti-allergic agents, anti-nausea agents (or anti-emetics), painrelievers, cytoprotective agents, and combinations thereof.

In one embodiment, a CoStAR-expressing cell described herein can be usedin combination with a chemotherapeutic agent. Exemplary chemotherapeuticagents include an anthracycline (e.g., doxorubicin (e.g., liposomaldoxorubicin)), a vinca alkaloid (e.g., vinblastine, vincristine,vindesine, vinorelbine), an alkylating agent (e.g., cyclophosphamide,decarbazine, melphalan, ifosfamide, temozolomide), an immune cellantibody (e.g., alemtuzamab, gemtuzumab, rituximab, ofatumumab,tositumomab, brentuximab), an antimetabolite (including, e.g., folicacid antagonists, pyrimidine analogs, purine analogs and adenosinedeaminase inhibitors (e.g., fludarabine)), an mTOR inhibitor, a TNFRglucocorticoid induced TNFR related protein (GITR) agonist, a proteasomeinhibitor (e.g., aclacinomycin A, gliotoxin or bortezomib), animmunomodulator such as thalidomide or a thalidomide derivative (e.g.,lenalidomide).

General Chemotherapeutic agents considered for use in combinationtherapies include busulfan (Myleran®), busulfan injection (Busulfex®),cladribine (Leustatin®), cyclophosphamide (Cytoxan® or Neosar®),cytarabine, cytosine arabinoside (Cytosar-U®), cytarabine liposomeinjection (DepoCyt®), daunorubicin hydrochloride (Cerubidine®),daunorubicin citrate liposome injection (DaunoXome®), dexamethasone,doxorubicin hydrochloride (Adriamycin®, Rubex®), etoposide (Vepesid®),fludarabine phosphate (Fludara®), hydroxyurea (Hydrea®), Idarubicin(Idamycin®), mitoxantrone (Novantrone®), Gemtuzumab Ozogamicin(Mylotarg®).

In embodiments, general chemotherapeutic agents considered for use incombination therapies include anastrozole (Arimidex®), bicalutamide(Casodex®), bleomycin sulfate (Blenoxane®), busulfan (Myleran®),busulfan injection (Busulfex®), capecitabine (Xeloda®),N4-pentoxycarbonyl-5-deoxy-5-fluorocytidine, carboplatin (Paraplatin®),carmustine (BiCNU®), chlorambucil (Leukeran®), cisplatin (Platinol®),cladribine (Leustatin®), cyclophosphamide (Cytoxan® or Neosar®),cytarabine, cytosine arabinoside (Cytosar-U®), cytarabine liposomeinjection (DepoCyt®), dacarbazine (DTIC—Dome®), dactinomycin(Actinomycin D, Cosmegan), daunorubicin hydrochloride (Cerubidine®),daunorubicin citrate liposome injection (DaunoXome®), dexamethasone,docetaxel (Taxotere®), doxorubicin hydrochloride (Adriamycin®, Rubex®),etoposide (Vepesid®), fludarabine phosphate (Fludara®), 5-fluorouracil(Adrucil®, Efudex®), flutamide (Eulexin®), tezacitibine, Gemcitabine(difluorodeoxycitidine), hydroxyurea (Hydrea®), Idarubicin (Idamycin®),ifosfamide (IFEX®), irinotecan (Camptosar®), L-asparaginase (ELSPAR®),leucovorin calcium, melphalan (Alkeran®), 6-mercaptopurine(Purinethol®), methotrexate (Folex®), mitoxantrone (Novantrone®),mylotarg, paclitaxel (Taxol®), phoenix (Yttrium90/MX-DTPA), pentostatin,polifeprosan 20 with carmustine implant (Gliadel®), tamoxifen citrate(Nolvadex®), teniposide (Vumon®), 6-thioguanine, thiotepa, tirapazamine(Tirazone®), topotecan hydrochloride for injection (Hycamptin®),vinblastine (Velban®), vincristine (Oncovin®), and vinorelbine(Navelbine®).

Treatments can be evaluated, for example, by tumor regression, tumorweight or size shrinkage, time to progression, duration of survival,progression free survival, overall response rate, duration of response,quality of life, protein expression and/or activity. Approaches todetermining efficacy of the therapy can be employed, including forexample, measurement of response through radiological imaging.

In some embodiments, any one of the sequences used or provided in Table7 and/or FIGS. 83 and 84 can be employed in the methods provided herein.In FIGS. 83 and 84 , the underlined bolded regions comprise CDRs. Insome embodiments, the antigen binding domain of the fusion protein cantarget CEA. In some embodiments, the antigen binding domain of thefusion protein can target CEA. In some embodiments the antigen bindingdomain of the fusion protein can target MSLN. In some embodiments, thisincludes some part of SEQ ID NO: 12 and/or parts of SEQ ID NO: 186-191,and/or variants thereof. In some embodiments, the fusion protein orCoSTaR comprises the MSLN construct components provided in FIG. 84 (allor in part or variants thereof). In some embodiments, this includes SEQID Nos: 192, 218, 228, 246, 264, or 282 (all or in part or variantsthereof).

Sequences

The following sequences include complete CoStARs and CoStAR componentsand are non-limiting. Components include signal peptides (SP), bindingdomains (BD), linkers, spacers and transmembrane domains (STM), a CD28transmembrane fragment without extracellular or intracellular sequences(STM-CD28TM), intracellular signal domains (SD) and CD40 domains andmotifs. Whereas SEQ ID NOS:42-247 or 196-335, 362-363, 369-420, and 514comprise CoStARs with N-terminal signal peptides, it will be understoodthat the N-terminal signal peptides are removed from a mature CoStAR.Further, is will be understood that a mature CoStAR may may lack 1, 2,3, 4, 5, 6, 7, 8, 9, or 10 or more amino acids at the N-terminal (i.e.,counted from the C-terminal end of the signal peptide). Componentlocations within whole proteins can be confirmed from exemplarysequences presented herein and from GenBank and other sources. Theconstructs and components are illustrative as to precise sizes andextents and components can be from more than one source. Where there ismore than one intracellular signaling domain or signaling fragment, themultiple domains can be in any order. It will be understood that whereascertain proteins may comprise N-terminal signal peptides when expressed,those signal peptides are cleaved and may be imprecisely cleaved whenthe proteins are expressed, and that the resulting proteins from whichsignal peptides are removed comprise binding domains having variation ofup to about five amino acids in the location of the N-terminal aminoacid.

TABLE 7 Amino Acid Sequences ID No Component Sequence 1 SP-OSMMGVLLTQRTL LSLVLALLFP SMASM 2 SP-CD8α MALPVTALLL PLALLLHAAR P 3 SP-CD2MSFPCKFVAS FLLIFNVSSK GAVS 4 SP-IL2 MYRMQLLSCI ALSLALVTNS 5 SP-GM-CSFMWLQSLLLLG TVACSIS 6 SP-hIgGk- MEAPAQLLFL LLLWLPDTTR VIII 7 SP-PD1MGVLLTQRTL LSLVLALLFP SMASM 8 SP-TIGIT MQIPQAPWPV VWAVLQLGWR PGW 9BD1-MOV QVQLQQSGAE LVKPGASVKI SCKASGYSFT GYFMNWVKQS HGKSLEWIGRIHPYDGDTFY NQNFKDKATL TVDKSSNTAH MELLSLTSED FAVYYCTRYDGSRAMDYWGQ GTTVTVSSGG GGSGGGGSGG GGSDIELTQS PASLAVSLGQRAIISCKASQ SVSFAGTSLM HWYHQKPGQQ PKLLIYRASN LEAGVPTRFSGSGSKTDFTL NIHPVEEEDA ATYYCQQSRE YPYTFGGGTK LEIK 10 MFE23QVKLQQSGAE LVRSGTSVKL SCTASGFNIK DSYMHWLRQG PEQGLEWIGWIDPENGDTEY APKFQGKATF TTDTSSNTAY LQLSSLTSED TAVYYCNEGTPTGPYYFDYW GQGTTVTVSS GGGGSGGGGS GGGGSENVLT QSPAIMSASPGEKVTITCSA SSSVSYMHWF QQKPGTSPKL WIYSTSNLAS GVPARFSGSGSGTSYSLTIS RMEAEDAATY YCQQRSSYPL TFGAGTKLEL KR 11 MFE23QVQLQQSGAE LVRSGTSVKL SCTASGFNIK DSYMHWLRQG PEQGLEWIGW (K > Q)IDPENGDTEY APKFQGKATF TTDTSSNTAY LQLSSLTSED TAVYYCNEGTPTGPYYFDYW GQGTTVTVSS GGGGSGGGGS GGGGSENVLT QSPAIMSASPGEKVTITCSA SSSVSYMHWF QQKPGTSPKL WIYSTSNLAS GVPARFSGSGSGTSYSLTIS RMEAEDAATY YCQQRSSYPL TFGAGTKLEL KR 12 hMFE23QVKLEQSGAE VVKPGASVKL SCKASGFNIK DSYMHWLRQG PGQRLEWIGWIDPENGDTEY APKFQGKATF TTDTSANTAY LGLSSLRPED TAVYYCNEGTPTGPYYFDYW GQGTLVTVSS GGGGSGGGGS GGGGSENVLT QSPSSMSASVGDRVNIACSA SSSVSYMHWF QQKPGKSPKL WIYSTSNLAS GVPSRFSGSGSGTDYSLTIS SMQPEDAATY YCQQRSSYPL TFGGGTKLEI K 13 CEA6QVQLVQSGAE VKKPGSSVKV SCKASGGTFS NSPINWLRQA PGQGLEWMGSIIPSFGTANY AQKFQGRLTI TADESTSTAY MELSSLRSED TAVYYCAGRSHNYELYYYYM DVWGQGTMVT VSSGGGGSGG GGSGGGGSDI QMTQSPSTLSASIGDRVTIT CRASEGIYHW LAWYQQKPGK APKLLIYKAS SLASGAPSRFSGSGSGTDFT LTISSLQPDD FATYYCQQYS NYPLTFGGGT KLEIKR 14 BW431/26QLQESGPGLV RPSQTLSLTC TVSGFTISSG YSWHWVRQPP GRGLEWIGYIQYSGITNYNP SLKSRVTMLV DTSKNQFSLR LSSVTAADTA VYYCAREDYDYHWYFDVWGQ GSLVTVSSGG GGSGGGGSGG GGSGVHSDIQ MTQSPSSLSASVGDRVTITC STSSSVSYMH WYQQKPGKAP KLLIYSTSNL ASGVPSRFSGSGSGTDFTFT ISSLQPEDIA TYYCHQWSSY PTFGQGTKVE IKR 15 HuT84.66EVQLVESGGG LVQPGGSLRL SCAASGFNIK DTYMHWVRQA PGKGLEWVAR (M5A)IDPANGNSKY ADSVKGRFTI SADTSKNTAY LQMNSLRAED TAVYYCAPFGYYVSDYAMAY WGQGTLVTVS SGGGGSGGGG SGGGGSDIQL TQSPSSLSASVGDRVTITCR AGESVDIFGV GFLHWYQQKP GKAPKLLIYR ASNLESGVPSRFSGSGSRTD FTLTISSLQP EDFATYYCQQ TNEDPYTFGQ GTKVEIK 16 BD1-PD1RPGWFLDSPD RPWNPPTFSP ALLVVTEGDN ATFTCSFSNT SESFVLNWYRMSPSNQTDKL AAFPEDRSQP GQDCRFRVTQ LPNGRDFHMS VVRARRNDSGTYLCGAISLA PKAQIKESLR AELRVTERRA EVPTAH 17 BD1-TIGITMMTGTIETTG NISAEKGGSI ILQCHLSSTT AQVTQVNWEQ QDQLLAICNADLGWHISPSF KDRVAPGPGL GLTLQSLTVN DTGEYFCIYH TYPDGTYTGRIFLEVLESSV AEHGARFQIP 18 3XA2Xgsg AAAGSGGSG 19 STM-ILVKQSPMLV AYDNAVNLSC KYSYNLFSRE FRASLHKGLD SAVEVCVVYG spCD28NYSQQLQVYS KTGFNCDGKL GNESVTFYLQ NLYVNQTDIY FCKIEVMYPPPYLDNEKSNG TIIHVKGKHL CPSPLFPGPS KPFWVLVVVG GVLACYSLLV TVAFIIFWV 20STM-spCD8 FVPVFLPAKP TTTPAPRPPT PAPTIASQPL SLRPEACRPA AGGAVHTRGLDFACDIYIWA PLAGTCGVLL LSLVITLYCN HRN 21 STM-FWVLVVVGGV LACYSLLVTV AFIIFWV CD28TM 22 STM-CPSPLFPGPS KPFWVLVVVG GVLACYSLLV TVAFIIFWV spCD28TM 23 STM-IIHVKGKHLC PSPLFPGPSK PFWVLVVVGG VLACYSLLVT VAFIIFWV CD28IIH(t run) 24STM-spIG4 ESKYGPPCPS CPAPEFLGGP SVFLFPPKPK DTLMISRTPE VTCVVVDVSQEDPEVQFNWY VDGVEVHNAK TKPREEQFNS TYRVVSVLTV LHQDWLNGKEYKCKVSNKGL PSSIEKTISK AKGQPREPQV YTLPPSQEEM TKNQVSLTCLVKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS RLTVDKSRWQEGNVFSCSVM HEALHNHYTQ KSLSLSLGKM FWVLVVVGGV LACYSLLVTV AFIIFWV 25Sig-CD28 RSKRSRLLHS DYMNMTPRRP GPTRKHYQPY APPRDFAAYR S 26 Sig-CD137RFSVVKRGRK KLLYIFKQPF MRPVQTTQEE DGCSCRFPEE EEGGCE 27 Sig-CD134RRDQRLPPDA HKPPGGGSFR TPIQEEQADA HSTLAKI 28 Sig-CD2KRKKQRSRRN DEELETRAHR VATEERGRKP HQIPASTPQN PATSQHPPPPPGHRSQAPSH RPPPPGHRVQ HQPQKRPPAP SGTQVHQQKG PPLPRPRVQP KPPHGAAENS LSPSSN29 Sig GITR QLGLHIWQLR SQCMWPRETQ LLLEVPPSTE DARSCQFPEE ERGERSAEEKGRLGDLWV 30 Sig-CD29 KLLMIIHDRR EFAKFEKEKM NAKWDTGENP IYKSAVTTVV NPKYEGK31 Sig-CD150 RRRGKTNHYQ TTVEKKSLTI YAQVQKPGPL QKKLDSFPAQ DPCTTIYVAATEPVPESVQE TNSITVYASV TLPES 32 CD40KKVAKKPTNK APHPKQEPQE INFPDDLPGS NTAAPVQETL HGCQPVTQED GKESRISVQE RQ 33CD40_ AKKPTNKAPH PKQEPQEINF PDDLPGSNTA APVQETLHGC QPVTQEDGKE tandemSRISVQERQK KVAKKPTNKA PHPKQEPQEI NFPDDLPGSN TAAPVQETLHGCQPVTQEDG KESRISVQER QKKVA 34 CD40_P227KKVAKKPTNK AAHPKQEPQE INFPDDLPGS NTAAPVQETL HGCQPVTQED A GKESRISVQE RQ35 SH3_motif KPTNKAPH 36 TRAF2_mot PKQE if1 37 TRAF2_mot PVQE if2 38TRAF2_mot SVQE if3 39 TRAF6_mot QEPQEINFP if 40 PKA_motif KKPTNKA 1 41PKA_motif SRISVQE 2 42 OSM_MFE23MGVLLTQRTL LSLVLALLFP SMASMQVKLQ QSGAELVRSG TSVKLSCTAS _spCD28GFNIKDSYMH WLRQGPEQGL EWIGWIDPEN GDTEYAPKFQ GKATFTTDTS _CD28_SNTAYLQLSS LTSEDTAVYY CNEGTPTGPY YFDYWGQGTT VTVSSGGGGS CD40GGGGSGGGGS ENVLTQSPAI MSASPGEKVT ITCSASSSVS YMHWFQQKPG CTP194TSPKLWIYST SNLASGVPAR FSGSGSGTSY SLTISRMEAE DAATYYCQQRSSYPLTFGAG TKLELKRAAA GSGGSGILVK QSPMLVAYDN AVNLSCKYSYNLFSREFRAS LHKGLDSAVE VCVVYGNYSQ QLQVYSKTGF NCDGKLGNESVTFYLQNLYV NQTDIYFCKI EVMYPPPYLD NEKSNGTIIH VKGKHLCPSPLFPGPSKPFW VLVVVGGVLA CYSLLVTVAF IIFWVRSKRS RLLHSDYMNMTPRRPGPTRK HYQPYAPPRD FAAYRSKKVA KKPTNKAPHP KQEPQEINFPDDLPGSNTAA PVQETLHGCQ PVTQEDGKES RISVQERQ 43 CD8α_MFE2MALPVTALLL PLALLLHAAR PQVKLQQSGA ELVRSGTSVK LSCTASGFNI 3KDSYMHWLRQ GPEQGLEWIG WIDPENGDTE YAPKFQGKAT FTTDTSSNTA _spCD28YLQLSSLTSE DTAVYYCNEG TPTGPYYFDY WGQGTTVTVS SGGGGSGGGG _CD28_SGGGGSENVL TQSPAIMSAS PGEKVTITCS ASSSVSYMHW FQQKPGTSPK CD40LWIYSTSNLA SGVPARFSGS GSGTSYSLTI SRMEAEDAAT YYCQQRSSYP CTP255LTFGAGTKLE LKRAAAGSGG SGILVKQSPM LVAYDNAVNL SCKYSYNLFSREFRASLHKG LDSAVEVCVV YGNYSQQLQV YSKTGFNCDG KLGNESVTFYLQNLYVNQTD IYFCKIEVMY PPPYLDNEKS NGTIIHVKGK HLCPSPLFPGPSKPFWVLVV VGGVLACYSL LVTVAFIIFW VRSKRSRLLH SDYMNMTPRRPGPTRKHYQP YAPPRDFAAY RSKKVAKKPT NKAPHPKQEP QEINFPDDLPGSNTAAPVQE TLHGCQPVTQ EDGKESRISV QERQ 44 CD2_MFE23MSFPCKFVAS FLLIFNVSSK GAVSQVKLQQ SGAELVRSGT SVKLSCTASG _spCD28FNIKDSYMHW LRQGPEQGLE WIGWIDPENG DTEYAPKFQG KATFTTDTSS _CD28_NTAYLQLSSL TSEDTAVYYC NEGTPTGPYY FDYWGQGTTV TVSSGGGGSG CD40GGGSGGGGSE NVLTQSPAIM SASPGEKVTI TCSASSSVSY MHWFQQKPGT CTP256SPKLWIYSTS NLASGVPARF SGSGSGTSYS LTISRMEAED AATYYCQQRSSYPLTFGAGT KLELKRAAAG SGGSGILVKQ SPMLVAYDNA VNLSCKYSYNLFSREFRASL HKGLDSAVEV CVVYGNYSQQ LQVYSKTGFN CDGKLGNESVTFYLQNLYVN QTDIYFCKIE VMYPPPYLDN EKSNGTIIHV KGKHLCPSPLFPGPSKPFWV LVVVGGVLAC YSLLVTVAFI IFWVRSKRSR LLHSDYMNMTPRRPGPTRKH YQPYAPPRDF AAYRSKKVAK KPTNKAPHPK QEPQEINFPDDLPGSNTAAP VQETLHGCQP VTQEDGKESR ISVQERQ 45 IL2_MFE23MYRMQLLSCI ALSLALVTNS QVKLQQSGAE LVRSGTSVKL SCTASGFNIK _spCD28DSYMHWLRQG PEQGLEWIGW IDPENGDTEY APKFQGKATF TTDTSSNTAY _CD28_LQLSSLTSED TAVYYCNEGT PTGPYYFDYW GQGTTVTVSS GGGGSGGGGS CD40GGGGSENVLT QSPAIMSASP GEKVTITCSA SSSVSYMHWF QQKPGTSPKL CTP257WIYSTSNLAS GVPARFSGSG SGTSYSLTIS RMEAEDAATY YCQQRSSYPLTFGAGTKLEL KRAAAGSGGS GILVKQSPML VAYDNAVNLS CKYSYNLFSREFRASLHKGL DSAVEVCVVY GNYSQQLQVY SKTGFNCDGK LGNESVTFYLQNLYVNQTDI YFCKIEVMYP PPYLDNEKSN GTIIHVKGKH LCPSPLFPGPSKPFWVLVVV GGVLACYSLL VTVAFIIFWV RSKRSRLLHS DYMNMTPRRPGPTRKHYQPY APPRDFAAYR SKKVAKKPTN KAPHPKQEPQ EINFPDDLPGSNTAAPVQET LHGCQPVTQE DGKESRISVQ ERQ 46 GM-MWLQSLLLLG TVACSISQVK LQQSGAELVR SGTSVKLSCT ASGFNIKDSY CSF_MFE23MHWLRQGPEQ GLEWIGWIDP ENGDTEYAPK FQGKATFTTD TSSNTAYLQL _spCD28SSLTSEDTAV YYCNEGTPTG PYYFDYWGQG TTVTVSSGGG GSGGGGSGGG _CD28_GSENVLTQSP AIMSASPGEK VTITCSASSS VSYMHWFQQK PGTSPKLWIY CD40STSNLASGVP ARFSGSGSGT SYSLTISRME AEDAATYYCQ QRSSYPLTFG CTP258AGTKLELKRA AAGSGGSGIL VKQSPMLVAY DNAVNLSCKY SYNLFSREFRASLHKGLDSA VEVCVVYGNY SQQLQVYSKT GFNCDGKLGN ESVTFYLQNLYVNQTDIYFC KIEVMYPPPY LDNEKSNGTI IHVKGKHLCP SPLFPGPSKPFWVLVVVGGV LACYSLLVTV AFIIFWVRSK RSRLLHSDYM NMTPRRPGPTRKHYQPYAPP RDFAAYRSKK VAKKPTNKAP HPKQEPQEIN FPDDLPGSNTAAPVQETLHG CQPVTQEDGK ESRISVQERQ 47 hIgGk-MEAPAQLLFL LLLWLPDTTR QVKLQQSGAE LVRSGTSVKL SCTASGFNIK VIII_MFE2DSYMHWLRQG PEQGLEWIGW IDPENGDTEY APKFQGKATF TTDTSSNTAY 3LQLSSLTSED TAVYYCNEGT PTGPYYFDYW GQGTTVTVSS GGGGSGGGGS _spCD28GGGGSENVLT QSPAIMSASP GEKVTITCSA SSSVSYMHWF QQKPGTSPKL _CD28_WIYSTSNLAS GVPARFSGSG SGTSYSLTIS RMEAEDAATY YCQQRSSYPL CD40TFGAGTKLEL KRAAAGSGGS GILVKQSPML VAYDNAVNLS CKYSYNLFSR CTP259EFRASLHKGL DSAVEVCVVY GNYSQQLQVY SKTGFNCDGK LGNESVTFYLQNLYVNQTDI YFCKIEVMYP PPYLDNEKSN GTIIHVKGKH LCPSPLFPGPSKPFWVLVVV GGVLACYSLL VTVAFIIFWV RSKRSRLLHS DYMNMTPRRPGPTRKHYQPY APPRDFAAYR SKKVAKKPTN KAPHPKQEPQ EINFPDDLPGSNTAAPVQET LHGCQPVTQE DGKESRISVQ ERQ 48 OSM_MFE23MGVLLTQRTL LSLVLALLFP SMASMQVKLQ QSGAELVRSG TSVKLSCTAS _spCD8GFNIKDSYMH WLRQGPEQGL EWIGWIDPEN GDTEYAPKFQ GKATFTTDTS _CD28_SNTAYLQLSS LTSEDTAVYY CNEGTPTGPY YFDYWGQGTT VTVSSGGGGS CD40GGGGSGGGGS ENVLTQSPAI MSASPGEKVT ITCSASSSVS YMHWFQQKPG CTP190TSPKLWIYST SNLASGVPAR FSGSGSGTSY SLTISRMEAE DAATYYCQQRSSYPLTFGAG TKLELKRAAA GSGGSGFVPV FLPAKPTTTP APRPPTPAPTIASQPLSLRP EACRPAAGGA VHTRGLDFAC DIYIWAPLAG TCGVLLLSLVITLYCNHRNR SKRSRLLHSD YMNMTPRRPG PTRKHYQPYA PPRDFAAYRSKKVAKKPTNK APHPKQEPQE INFPDDLPGS NTAAPVQETL HGCQPVTQED GKESRISVQE RQ 49CD8a_MFE2 MALPVTALLL PLALLLHAAR PQVKLQQSGA ELVRSGTSVK LSCTASGFNI 3KDSYMHWLRQ GPEQGLEWIG WIDPENGDTE YAPKFQGKAT FTTDTSSNTA _spCD8YLQLSSLTSE DTAVYYCNEG TPTGPYYFDY WGQGTTVTVS SGGGGSGGGG _CD28_SGGGGSENVL TQSPAIMSAS PGEKVTITCS ASSSVSYMHW FQQKPGTSPK CD40LWIYSTSNLA SGVPARFSGS GSGTSYSLTI SRMEAEDAAT YYCQQRSSYPLTFGAGTKLE LKRAAAGSGG SGFVPVFLPA KPTTTPAPRP PTPAPTIASQPLSLRPEACR PAAGGAVHTR GLDFACDIYI WAPLAGTCGV LLLSLVITLYCNHRNRSKRS RLLHSDYMNM TPRRPGPTRK HYQPYAPPRD FAAYRSKKVAKKPTNKAPHP KQEPQEINFP DDLPGSNTAA PVQETLHGCQ PVTQEDGKES RISVQERQ 50CD2_MFE23 MSFPCKFVAS FLLIFNVSSK GAVSQVKLQQ SGAELVRSGT SVKLSCTASG _spCD8FNIKDSYMHW LRQGPEQGLE WIGWIDPENG DTEYAPKFQG KATFTTDTSS _CD28_NTAYLQLSSL TSEDTAVYYC NEGTPTGPYY FDYWGQGTTV TVSSGGGGSG CD40GGGSGGGGSE NVLTQSPAIM SASPGEKVTI TCSASSSVSY MHWFQQKPGTSPKLWIYSTS NLASGVPARF SGSGSGTSYS LTISRMEAED AATYYCQQRSSYPLTFGAGT KLELKRAAAG SGGSGFVPVF LPAKPTTTPA PRPPTPAPTIASQPLSLRPE ACRPAAGGAV HTRGLDFACD IYIWAPLAGT CGVLLLSLVITLYCNHRNRS KRSRLLHSDY MNMTPRRPGP TRKHYQPYAP PRDFAAYRSKKVAKKPTNKA PHPKQEPQEI NFPDDLPGSN TAAPVQETLH GCQPVTQEDG KESRISVQER Q 51IL2_MFE23 MYRMQLLSCI ALSLALVTNS QVKLQQSGAE LVRSGTSVKL SCTASGFNIK _spCD8DSYMHWLRQG PEQGLEWIGW IDPENGDTEY APKFQGKATF TTDTSSNTAY _CD28_LQLSSLTSED TAVYYCNEGT PTGPYYFDYW GQGTTVTVSS GGGGSGGGGS CD40GGGGSENVLT QSPAIMSASP GEKVTITCSA SSSVSYMHWF QQKPGTSPKLWIYSTSNLAS GVPARFSGSG SGTSYSLTIS RMEAEDAATY YCQQRSSYPLTFGAGTKLEL KRAAAGSGGS GFVPVFLPAK PTTTPAPRPP TPAPTIASQPLSLRPEACRP AAGGAVHTRG LDFACDIYIW APLAGTCGVL LLSLVITLYCNHRNRSKRSR LLHSDYMNMT PRRPGPTRKH YQPYAPPRDF AAYRSKKVAKKPTNKAPHPK QEPQEINFPD DLPGSNTAAP VQETLHGCQP VTQEDGKESR ISVQERQ 52 GM-MWLQSLLLLG TVACSISQVK LQQSGAELVR SGTSVKLSCT ASGFNIKDSY CSF_MFE23MHWLRQGPEQ GLEWIGWIDP ENGDTEYAPK FQGKATFTTD TSSNTAYLQL _spCD8SSLTSEDTAV YYCNEGTPTG PYYFDYWGQG TTVTVSSGGG GSGGGGSGGG _CD28_GSENVLTQSP AIMSASPGEK VTITCSASSS VSYMHWFQQK PGTSPKLWIY CD40STSNLASGVP ARFSGSGSGT SYSLTISRME AEDAATYYCQ QRSSYPLTFGAGTKLELKRA AAGSGGSGFV PVFLPAKPTT TPAPRPPTPA PTIASQPLSLRPEACRPAAG GAVHTRGLDF ACDIYIWAPL AGTCGVLLLS LVITLYCNHRNRSKRSRLLH SDYMNMTPRR PGPTRKHYQP YAPPRDFAAY RSKKVAKKPTNKAPHPKQEP QEINFPDDLP GSNTAAPVQE TLHGCQPVTQ EDGKESRISV QERQ 53 hIgGk-MEAPAQLLFL LLLWLPDTTR QVKLQQSGAE LVRSGTSVKL SCTASGFNIK VIII_MFE2DSYMHWLRQG PEQGLEWIGW IDPENGDTEY APKFQGKATF TTDTSSNTAY 3LQLSSLTSED TAVYYCNEGT PTGPYYFDYW GQGTTVTVSS GGGGSGGGGS _spCD8GGGGSENVLT QSPAIMSASP GEKVTITCSA SSSVSYMHWF QQKPGTSPKL _CD28_WIYSTSNLAS GVPARFSGSG SGTSYSLTIS RMEAEDAATY YCQQRSSYPL CD40TFGAGTKLEL KRAAAGSGGS GFVPVFLPAK PTTTPAPRPP TPAPTIASQPLSLRPEACRP AAGGAVHTRG LDFACDIYIW APLAGTCGVL LLSLVITLYCNHRNRSKRSR LLHSDYMNMT PRRPGPTRKH YQPYAPPRDF AAYRSKKVAKKPTNKAPHPK QEPQEINFPD DLPGSNTAAP VQETLHGCQP VTQEDGKESR ISVQERQ 54OSM_MFE23 MGVLLTQRTL LSLVLALLFP SMASMQVKLQ QSGAELVRSG TSVKLSCTAS _CD28TMGFNIKDSYMH WLRQGPEQGL EWIGWIDPEN GDTEYAPKFQ GKATFTTDTS _CD28_SNTAYLQLSS LTSEDTAVYY CNEGTPTGPY YFDYWGQGTT VTVSSGGGGS CD40GGGGSGGGGS ENVLTQSPAI MSASPGEKVT ITCSASSSVS YMHWFQQKPGTSPKLWIYST SNLASGVPAR FSGSGSGTSY SLTISRMEAE DAATYYCQQRSSYPLTFGAG TKLELKRAAA GSGGSGFWVL VVVGGVLACY SLLVTVAFIIFWVRSKRSRL LHSDYMNMTP RRPGPTRKHY QPYAPPRDFA AYRSKKVAKKPTNKAPHPKQ EPQEINFPDD LPGSNTAAPV QETLHGCQPV TQEDGKESRI SVQERQ 55CD8a_MFE2 MALPVTALLL PLALLLHAAR PQVKLQQSGA ELVRSGTSVK LSCTASGFNI 3KDSYMHWLRQ GPEQGLEWIG WIDPENGDTE YAPKFQGKAT FTTDTSSNTA _CD28TMYLQLSSLTSE DTAVYYCNEG TPTGPYYFDY WGQGTTVTVS SGGGGSGGGG _CD28_SGGGGSENVL TQSPAIMSAS PGEKVTITCS ASSSVSYMHW FQQKPGTSPK CD40LWIYSTSNLA SGVPARFSGS GSGTSYSLTI SRMEAEDAAT YYCQQRSSYPLTFGAGTKLE LKRAAAGSGG SGFWVLVVVG GVLACYSLLV TVAFIIFWVRSKRSRLLHSD YMNMTPRRPG PTRKHYQPYA PPRDFAAYRS KKVAKKPTNKAPHPKQEPQE INFPDDLPGS NTAAPVQETL HGCQPVTQED GKESRISVQE RQ 56 CD2_MFE23MSFPCKFVAS FLLIFNVSSK GAVSQVKLQQ SGAELVRSGT SVKLSCTASG _CD28TMFNIKDSYMHW LRQGPEQGLE WIGWIDPENG DTEYAPKFQG KATFTTDTSS _CD28_NTAYLQLSSL TSEDTAVYYC NEGTPTGPYY FDYWGQGTTV TVSSGGGGSG CD40GGGSGGGGSE NVLTQSPAIM SASPGEKVTI TCSASSSVSY MHWFQQKPGTSPKLWIYSTS NLASGVPARF SGSGSGTSYS LTISRMEAED AATYYCQQRSSYPLTFGAGT KLELKRAAAG SGGSGFWVLV VVGGVLACYS LLVTVAFIIFWVRSKRSRLL HSDYMNMTPR RPGPTRKHYQ PYAPPRDFAA YRSKKVAKKPTNKAPHPKQE PQEINFPDDL PGSNTAAPVQ ETLHGCQPVT QEDGKESRIS VQERQ 57IL2_MFE23 MYRMQLLSCI ALSLALVTNS QVKLQQSGAE LVRSGTSVKL SCTASGFNIK _CD28TMDSYMHWLRQG PEQGLEWIGW IDPENGDTEY APKFQGKATF TTDTSSNTAY _CD28_LQLSSLTSED TAVYYCNEGT PTGPYYFDYW GQGTTVTVSS GGGGSGGGGS CD40GGGGSENVLT QSPAIMSASP GEKVTITCSA SSSVSYMHWF QQKPGTSPKLWIYSTSNLAS GVPARFSGSG SGTSYSLTIS RMEAEDAATY YCQQRSSYPLTFGAGTKLEL KRAAAGSGGS GFWVLVVVGG VLACYSLLVT VAFIIFWVRSKRSRLLHSDY MNMTPRRPGP TRKHYQPYAP PRDFAAYRSK KVAKKPTNKAPHPKQEPQEI NFPDDLPGSN TAAPVQETLH GCQPVTQEDG KESRISVQER Q 58 GM-MWLQSLLLLG TVACSISQVK LQQSGAELVR SGTSVKLSCT ASGFNIKDSY CSF_MFE23MHWLRQGPEQ GLEWIGWIDP ENGDTEYAPK FQGKATFTTD TSSNTAYLQL _CD28TMSSLTSEDTAV YYCNEGTPTG PYYFDYWGQG TTVTVSSGGG GSGGGGSGGG _CD28_GSENVLTQSP AIMSASPGEK VTITCSASSS VSYMHWFQQK PGTSPKLWIY CD40STSNLASGVP ARFSGSGSGT SYSLTISRME AEDAATYYCQ QRSSYPLTFGAGTKLELKRA AAGSGGSGFW VLVVVGGVLA CYSLLVTVAF IIFWVRSKRSRLLHSDYMNM TPRRPGPTRK HYQPYAPPRD FAAYRSKKVA KKPTNKAPHPKQEPQEINFP DDLPGSNTAA PVQETLHGCQ PVTQEDGKES RISVQERQ 59 hIgGk-MEAPAQLLFL LLLWLPDTTR QVKLQQSGAE LVRSGTSVKL SCTASGFNIK VIII_MFE2DSYMHWLRQG PEQGLEWIGW IDPENGDTEY APKFQGKATF TTDTSSNTAY 3LQLSSLTSED TAVYYCNEGT PTGPYYFDYW GQGTTVTVSS GGGGSGGGGS _CD28TMGGGGSENVLT QSPAIMSASP GEKVTITCSA SSSVSYMHWF QQKPGTSPKL _CD28_WIYSTSNLAS GVPARFSGSG SGTSYSLTIS RMEAEDAATY YCQQRSSYPL CD40TFGAGTKLEL KRAAAGSGGS GFWVLVVVGG VLACYSLLVT VAFIIFWVRSKRSRLLHSDY MNMTPRRPGP TRKHYQPYAP PRDFAAYRSK KVAKKPTNKAPHPKQEPQEI NFPDDLPGSN TAAPVQETLH GCQPVTQEDG KESRISVQER Q 60 OSM_MFE23MGVLLTQRTL LSLVLALLFP SMASMQVQLQ QSGAELVRSG TSVKLSCTAS (K > Q)_spCGFNIKDSYMH WLRQGPEQGL EWIGWIDPEN GDTEYAPKFQ GKATFTTDTS D28SNTAYLQLSS LTSEDTAVYY CNEGTPTGPY YFDYWGQGTT VTVSSGGGGS _CD28_GGGGSGGGGS ENVLTQSPAI MSASPGEKVT ITCSASSSVS YMHWFQQKPG CD40TSPKLWIYST SNLASGVPAR FSGSGSGTSY SLTISRMEAE DAATYYCQQR CTP219SSYPLTFGAG TKLELKRAAA GSGGSGILVK QSPMLVAYDN AVNLSCKYSYNLFSREFRAS LHKGLDSAVE VCVVYGNYSQ QLQVYSKTGF NCDGKLGNESVTFYLQNLYV NQTDIYFCKI EVMYPPPYLD NEKSNGTIIH VKGKHLCPSPLFPGPSKPFW VLVVVGGVLA CYSLLVTVAF IIFWVRSKRS RLLHSDYMNMTPRRPGPTRK HYQPYAPPRD FAAYRSKKVA KKPTNKAPHP KQEPQEINFPDDLPGSNTAA PVQETLHGCQ PVTQEDGKES RISVQERQ 61 CD8a_MFE2MALPVTALLL PLALLLHAAR PQVQLQQSGA ELVRSGTSVK LSCTASGFNI 3KDSYMHWLRQ GPEQGLEWIG WIDPENGDTE YAPKFQGKAT FTTDTSSNTA (K > Q)_spCYLQLSSLTSE DTAVYYCNEG TPTGPYYFDY WGQGTTVTVS SGGGGSGGGG D28SGGGGSENVL TQSPAIMSAS PGEKVTITCS ASSSVSYMHW FQQKPGTSPK _CD28_LWIYSTSNLA SGVPARFSGS GSGTSYSLTI SRMEAEDAAT YYCQQRSSYP CD40LTFGAGTKLE LKRAAAGSGG SGILVKQSPM LVAYDNAVNL SCKYSYNLFSREFRASLHKG LDSAVEVCVV YGNYSQQLQV YSKTGFNCDG KLGNESVTFYLQNLYVNQTD IYFCKIEVMY PPPYLDNEKS NGTIIHVKGK HLCPSPLFPGPSKPFWVLVV VGGVLACYSL LVTVAFIIFW VRSKRSRLLH SDYMNMTPRRPGPTRKHYQP YAPPRDFAAY RSKKVAKKPT NKAPHPKQEP QEINFPDDLPGSNTAAPVQE TLHGCQPVTQ EDGKESRISV QERQ 62 CD2_MFE23MSFPCKFVAS FLLIFNVSSK GAVSQVQLQQ SGAELVRSGT SVKLSCTASG (K > Q)FNIKDSYMHW LRQGPEQGLE WIGWIDPENG DTEYAPKFQG KATFTTDTSS _spCD28NTAYLQLSSL TSEDTAVYYC NEGTPTGPYY FDYWGQGTTV TVSSGGGGSG _CD28_GGGSGGGGSE NVLTQSPAIM SASPGEKVTI TCSASSSVSY MHWFQQKPGT CD40SPKLWIYSTS NLASGVPARF SGSGSGTSYS LTISRMEAED AATYYCQQRSSYPLTFGAGT KLELKRAAAG SGGSGILVKQ SPMLVAYDNA VNLSCKYSYNLFSREFRASL HKGLDSAVEV CVVYGNYSQQ LQVYSKTGFN CDGKLGNESVTFYLQNLYVN QTDIYFCKIE VMYPPPYLDN EKSNGTIIHV KGKHLCPSPLFPGPSKPFWV LVVVGGVLAC YSLLVTVAFI IFWVRSKRSR LLHSDYMNMTPRRPGPTRKH YQPYAPPRDF AAYRSKKVAK KPTNKAPHPK QEPQEINFPDDLPGSNTAAP VQETLHGCQP VTQEDGKESR ISVQERQ 63 IL2_MFE23MYRMQLLSCI ALSLALVTNS QVQLQQSGAE LVRSGTSVKL SCTASGFNIK (K > Q)DSYMHWLRQG PEQGLEWIGW IDPENGDTEY APKFQGKATF TTDTSSNTAY _spCD28LQLSSLTSED TAVYYCNEGT PTGPYYFDYW GQGTTVTVSS GGGGSGGGGS _CD28_GGGGSENVLT QSPAIMSASP GEKVTITCSA SSSVSYMHWF QQKPGTSPKL CD40WIYSTSNLAS GVPARFSGSG SGTSYSLTIS RMEAEDAATY YCQQRSSYPLTFGAGTKLEL KRAAAGSGGS GILVKQSPML VAYDNAVNLS CKYSYNLFSREFRASLHKGL DSAVEVCVVY GNYSQQLQVY SKTGFNCDGK LGNESVTFYLQNLYVNQTDI YFCKIEVMYP PPYLDNEKSN GTIIHVKGKH LCPSPLFPGPSKPFWVLVVV GGVLACYSLL VTVAFIIFWV RSKRSRLLHS DYMNMTPRRPGPTRKHYQPY APPRDFAAYR SKKVAKKPTN KAPHPKQEPQ EINFPDDLPGSNTAAPVQET LHGCQPVTQE DGKESRISVQ ERQ 64 GM-CSFMWLQSLLLLG TVACSISQVQ LQQSGAELVR SGTSVKLSCT ASGFNIKDSY _MFE23MHWLRQGPEQ GLEWIGWIDP ENGDTEYAPK FQGKATFTTD TSSNTAYLQL (K > Q)SSLTSEDTAV YYCNEGTPTG PYYFDYWGQG TTVTVSSGGG GSGGGGSGGG _spCD28GSENVLTQSP AIMSASPGEK VTITCSASSS VSYMHWFQQK PGTSPKLWIY _CD28_STSNLASGVP ARFSGSGSGT SYSLTISRME AEDAATYYCQ QRSSYPLTFG CD40AGTKLELKRA AAGSGGSGIL VKQSPMLVAY DNAVNLSCKY SYNLFSREFRASLHKGLDSA VEVCVVYGNY SQQLQVYSKT GFNCDGKLGN ESVTFYLQNLYVNQTDIYFC KIEVMYPPPY LDNEKSNGTI IHVKGKHLCP SPLFPGPSKPFWVLVVVGGV LACYSLLVTV AFIIFWVRSK RSRLLHSDYM NMTPRRPGPTRKHYQPYAPP RDFAAYRSKK VAKKPTNKAP HPKQEPQEIN FPDDLPGSNTAAPVQETLHG CQPVTQEDGK ESRISVQERQ 65 hIgGk-MEAPAQLLFL LLLWLPDTTR QVQLQQSGAE LVRSGTSVKL SCTASGFNIK VIIIDSYMHWLRQG PEQGLEWIGW IDPENGDTEY APKFQGKATF TTDTSSNTAY _MFE23(K >LQLSSLTSED TAVYYCNEGT PTGPYYFDYW GQGTTVTVSS GGGGSGGGGS Q)GGGGSENVLT QSPAIMSASP GEKVTITCSA SSSVSYMHWF QQKPGTSPKL _spCD28WIYSTSNLAS GVPARFSGSG SGTSYSLTIS RMEAEDAATY YCQQRSSYPL _CD28_TFGAGTKLEL KRAAAGSGGS GILVKQSPML VAYDNAVNLS CKYSYNLFSR CD40EFRASLHKGL DSAVEVCVVY GNYSQQLQVY SKTGFNCDGK LGNESVTFYLQNLYVNQTDI YFCKIEVMYP PPYLDNEKSN GTIIHVKGKH LCPSPLFPGPSKPFWVLVVV GGVLACYSLL VTVAFIIFWV RSKRSRLLHS DYMNMTPRRPGPTRKHYQPY APPRDFAAYR SKKVAKKPTN KAPHPKQEPQ EINFPDDLPGSNTAAPVQET LHGCQPVTQE DGKESRISVQ ERQ 66 OSM_MFE23MGVLLTQRTL LSLVLALLFP SMASMQVOLQ QSGAELVRSG TSVKLSCTAS (K > Q)GFNIKDSYMH WLRQGPEQGL EWIGWIDPEN GDTEYAPKFQ GKATFTTDTS _spCD8SNTAYLQLSS LTSEDTAVYY CNEGTPTGPY YFDYWGQGTT VTVSSGGGGS _CD28_GGGGSGGGGS ENVLTQSPAI MSASPGEKVT ITCSASSSVS YMHWFQQKPG CD40TSPKLWIYST SNLASGVPAR FSGSGSGTSY SLTISRMEAE DAATYYCQQRSSYPLTFGAG TKLELKRAAA GSGGSGFVPV FLPAKPTTTP APRPPTPAPTIASQPLSLRP EACRPAAGGA VHTRGLDFAC DIYIWAPLAG TCGVLLLSLVITLYCNHRNR SKRSRLLHSD YMNMTPRRPG PTRKHYQPYA PPRDFAAYRSKKVAKKPTNK APHPKQEPQE INFPDDLPGS NTAAPVQETL HGCQPVTQED GKESRISVQE RQ 67CD8a MALPVTALLL PLALLLHAAR PQVQLQQSGA ELVRSGTSVK LSCTASGFNI _MFE2 3 (K >KDSYMHWLRQ GPEQGLEWIG WIDPENGDTE YAPKFQGKAT FTTDTSSNTA Q)YLQLSSLTSE DTAVYYCNEG TPTGPYYFDY WGQGTTVTVS SGGGGSGGGG _spCD8SGGGGSENVL TQSPAIMSAS PGEKVTITCS ASSSVSYMHW FQQKPGTSPK _CD28_LWIYSTSNLA SGVPARFSGS GSGTSYSLTI SRMEAEDAAT YYCQQRSSYP CD40LTFGAGTKLE LKRAAAGSGG SGFVPVFLPA KPTTTPAPRP PTPAPTIASQPLSLRPEACR PAAGGAVHTR GLDFACDIYI WAPLAGTCGV LLLSLVITLYCNHRNRSKRS RLLHSDYMNM TPRRPGPTRK HYQPYAPPRD FAAYRSKKVAKKPTNKAPHP KQEPQEINFP DDLPGSNTAA PVQETLHGCQ PVTQEDGKES RISVQERQ 68CD2_MFE23 MSFPCKFVAS FLLIFNVSSK GAVSQVQLQQ SGAELVRSGT SVKLSCTASG (K > Q)FNIKDSYMHW LRQGPEQGLE WIGWIDPENG DTEYAPKFQG KATFTTDTSS _spCD8NTAYLQLSSL TSEDTAVYYC NEGTPTGPYY FDYWGQGTTV TVSSGGGGSG _CD28_GGGSGGGGSE NVLTQSPAIM SASPGEKVTI TCSASSSVSY MHWFQQKPGT CD40SPKLWIYSTS NLASGVPARF SGSGSGTSYS LTISRMEAED AATYYCQQRSSYPLTFGAGT KLELKRAAAG SGGSGFVPVF LPAKPTTTPA PRPPTPAPTIASQPLSLRPE ACRPAAGGAV HTRGLDFACD IYIWAPLAGT CGVLLLSLVITLYCNHRNRS KRSRLLHSDY MNMTPRRPGP TRKHYQPYAP PRDFAAYRSKKVAKKPTNKA PHPKQEPQEI NFPDDLPGSN TAAPVQETLH GCQPVTQEDG KESRISVQER Q 69IL2_MFE23 MYRMQLLSCI ALSLALVTNS QVQLQQSGAE LVRSGTSVKL SCTASGFNIK (K > Q)DSYMHWLRQG PEQGLEWIGW IDPENGDTEY APKFQGKATF TTDTSSNTAY _spCD8LQLSSLTSED TAVYYCNEGT PTGPYYFDYW GQGTTVTVSS GGGGSGGGGS _CD28_GGGGSENVLT QSPAIMSASP GEKVTITCSA SSSVSYMHWF QQKPGTSPKL CD40WIYSTSNLAS GVPARFSGSG SGTSYSLTIS RMEAEDAATY YCQQRSSYPLTFGAGTKLEL KRAAAGSGGS GFVPVFLPAK PTTTPAPRPP TPAPTIASQPLSLRPEACRP AAGGAVHTRG LDFACDIYIW APLAGTCGVL LLSLVITLYCNHRNRSKRSR LLHSDYMNMT PRRPGPTRKH YQPYAPPRDF AAYRSKKVAKKPTNKAPHPK QEPQEINFPD DLPGSNTAAP VQETLHGCQP VTQEDGKESR ISVQERQ 70 GM-CSFMWLQSLLLLG TVACSISQVQ LQQSGAELVR SGTSVKLSCT ASGFNIKDSY _MFE23(K >MHWLRQGPEQ GLEWIGWIDP ENGDTEYAPK FQGKATFTTD TSSNTAYLQL Q)SSLTSEDTAV YYCNEGTPTG PYYFDYWGQG TTVTVSSGGG GSGGGGSGGG _spCD8GSENVLTQSP AIMSASPGEK VTITCSASSS VSYMHWFQQK PGTSPKLWIY _CD28_STSNLASGVP ARFSGSGSGT SYSLTISRME AEDAATYYCQ QRSSYPLTFG CD40AGTKLELKRA AAGSGGSGFV PVFLPAKPTT TPAPRPPTPA PTIASQPLSLRPEACRPAAG GAVHTRGLDF ACDIYIWAPL AGTCGVLLLS LVITLYCNHRNRSKRSRLLH SDYMNMTPRR PGPTRKHYQP YAPPRDFAAY RSKKVAKKPTNKAPHPKQEP QEINFPDDLP GSNTAAPVQE TLHGCQPVTQ EDGKESRISV QERQ 71 hIgGk-MEAPAQLLFL LLLWLPDTTR QVQLQQSGAE LVRSGTSVKL SCTASGFNIK VIIIDSYMHWLRQG PEQGLEWIGW IDPENGDTEY APKFQGKATF TTDTSSNTAY _MFE2 3 (K >LQLSSLTSED TAVYYCNEGT PTGPYYFDYW GQGTTVTVSS GGGGSGGGGS Q)GGGGSENVLT QSPAIMSASP GEKVTITCSA SSSVSYMHWF QQKPGTSPKL _spCD8WIYSTSNLAS GVPARFSGSG SGTSYSLTIS RMEAEDAATY YCQQRSSYPL _CD28_TFGAGTKLEL KRAAAGSGGS GFVPVFLPAK PTTTPAPRPP TPAPTIASQP CD40LSLRPEACRP AAGGAVHTRG LDFACDIYIW APLAGTCGVL LLSLVITLYCNHRNRSKRSR LLHSDYMNMT PRRPGPTRKH YQPYAPPRDF AAYRSKKVAKKPTNKAPHPK QEPQEINFPD DLPGSNTAAP VQETLHGCQP VTQEDGKESR ISVQERQ 72OSM_MFE23 MGVLLTQRTL LSLVLALLFP SMASMQVOLQ QSGAELVRSG TSVKLSCTAS (K > Q)GFNIKDSYMH WLRQGPEQGL EWIGWIDPEN GDTEYAPKFQ GKATFTTDTS _CD28TMSNTAYLQLSS LTSEDTAVYY CNEGTPTGPY YFDYWGQGTT VTVSSGGGGS _CD28_GGGGSGGGGS ENVLTQSPAI MSASPGEKVT ITCSASSSVS YMHWFQQKPG CD40TSPKLWIYST SNLASGVPAR FSGSGSGTSY SLTISRMEAE DAATYYCQQRSSYPLTFGAG TKLELKRAAA GSGGSGFWVL VVVGGVLACY SLLVTVAFIIFWVRSKRSRL LHSDYMNMTP RRPGPTRKHY QPYAPPRDFA AYRSKKVAKKPTNKAPHPKQ EPQEINFPDD LPGSNTAAPV QETLHGCQPV TQEDGKESRI SVQERQ 73 CD8aMALPVTALLL PLALLLHAAR PQVQLQQSGA ELVRSGTSVK LSCTASGFNI _MFE23(K >KDSYMHWLRQ GPEQGLEWIG WIDPENGDTE YAPKFQGKAT FTTDTSSNTA Q)YLQLSSLTSE DTAVYYCNEG TPTGPYYFDY WGQGTTVTVS SGGGGSGGGG _CD28TMSGGGGSENVL TQSPAIMSAS PGEKVTITCS ASSSVSYMHW FQQKPGTSPK _CD28_LWIYSTSNLA SGVPARFSGS GSGTSYSLTI SRMEAEDAAT YYCQQRSSYP CD40LTFGAGTKLE LKRAAAGSGG SGFWVLVVVG GVLACYSLLV TVAFIIFWVRSKRSRLLHSD YMNMTPRRPG PTRKHYQPYA PPRDFAAYRS KKVAKKPTNKAPHPKQEPQE INFPDDLPGS NTAAPVQETL HGCQPVTQED GKESRISVQE RQ 74 CD2_MFE23MSFPCKFVAS FLLIFNVSSK GAVSQVQLQQ SGAELVRSGT SVKLSCTASG (K > Q)FNIKDSYMHW LRQGPEQGLE WIGWIDPENG DTEYAPKFQG KATFTTDTSS _CD28TMNTAYLQLSSL TSEDTAVYYC NEGTPTGPYY FDYWGQGTTV TVSSGGGGSG _CD28_GGGSGGGGSE NVLTQSPAIM SASPGEKVTI TCSASSSVSY MHWFQQKPGT CD40SPKLWIYSTS NLASGVPARF SGSGSGTSYS LTISRMEAED AATYYCQQRSSYPLTFGAGT KLELKRAAAG SGGSGFWVLV VVGGVLACYS LLVTVAFIIFWVRSKRSRLL HSDYMNMTPR RPGPTRKHYQ PYAPPRDFAA YRSKKVAKKPTNKAPHPKQE PQEINFPDDL PGSNTAAPVQ ETLHGCQPVT QEDGKESRIS VQERQ 75IL2_MFE23 MYRMQLLSCI ALSLALVTNS QVQLQQSGAE LVRSGTSVKL SCTASGFNIK (K > Q)DSYMHWLRQG PEQGLEWIGW IDPENGDTEY APKFQGKATF TTDTSSNTAY _CD28TMLQLSSLTSED TAVYYCNEGT PTGPYYFDYW GQGTTVTVSS GGGGSGGGGS _CD28_GGGGSENVLT QSPAIMSASP GEKVTITCSA SSSVSYMHWF QQKPGTSPKL CD40WIYSTSNLAS GVPARFSGSG SGTSYSLTIS RMEAEDAATY YCQQRSSYPLTFGAGTKLEL KRAAAGSGGS GFWVLVVVGG VLACYSLLVT VAFIIFWVRSKRSRLLHSDY MNMTPRRPGP TRKHYQPYAP PRDFAAYRSK KVAKKPTNKAPHPKQEPQEI NFPDDLPGSN TAAPVQETLH GCQPVTQEDG KESRISVQER Q 76 GM-CSFMWLQSLLLLG TVACSISQVQ LQQSGAELVR SGTSVKLSCT ASGFNIKDSY _MFE23(K >MHWLRQGPEQ GLEWIGWIDP ENGDTEYAPK FQGKATFTTD TSSNTAYLQL Q)SSLTSEDTAV YYCNEGTPTG PYYFDYWGQG TTVTVSSGGG GSGGGGSGGG _CD28TMGSENVLTQSP AIMSASPGEK VTITCSASSS VSYMHWFQQK PGTSPKLWIY _CD28_STSNLASGVP ARFSGSGSGT SYSLTISRME AEDAATYYCQ QRSSYPLTFG CD40AGTKLELKRA AAGSGGSGFW VLVVVGGVLA CYSLLVTVAF IIFWVRSKRSRLLHSDYMNM TPRRPGPTRK HYQPYAPPRD FAAYRSKKVA KKPTNKAPHPKQEPQEINFP DDLPGSNTAA PVQETLHGCQ PVTQEDGKES RISVQERQ 77 hIgGk-MEAPAQLLFL LLLWLPDTTR QVQLQQSGAE LVRSGTSVKL SCTASGFNIK VIIIDSYMHWLRQG PEQGLEWIGW IDPENGDTEY APKFQGKATF TTDTSSNTAY _MFE23(K >LQLSSLTSED TAVYYCNEGT PTGPYYFDYW GQGTTVTVSS GGGGSGGGGS Q)GGGGSENVLT QSPAIMSASP GEKVTITCSA SSSVSYMHWF QQKPGTSPKL _CD28TMWIYSTSNLAS GVPARFSGSG SGTSYSLTIS RMEAEDAATY YCQQRSSYPL _CD28_TFGAGTKLEL KRAAAGSGGS GFWVLVVVGG VLACYSLLVT VAFIIFWVRS CD40KRSRLLHSDY MNMTPRRPGP TRKHYQPYAP PRDFAAYRSK KVAKKPTNKAPHPKQEPQEI NFPDDLPGSN TAAPVQETLH GCQPVTQEDG KESRISVQER Q 78 OSM_HuMFEMGVLLTQRTL LSLVLALLFP SMASMQVKLE QSGAEVVKPG ASVKLSCKAS 23GFNIKDSYMH WLRQGPGQRL EWIGWIDPEN GDTEYAPKFQ GKATFTTDTS _spCD28ANTAYLGLSS LRPEDTAVYY CNEGTPTGPY YFDYWGQGTL VTVSSGGGGS _CD28_GGGGSGGGGS ENVLTQSPSS MSASVGDRVN IACSASSSVS YMHWFQQKPG CD40KSPKLWIYST SNLASGVPSR FSGSGSGTDY SLTISSMQPE DAATYYCQQR CTP220SSYPLTFGGG TKLEIKAAAG SGGSGILVKQ SPMLVAYDNA VNLSCKYSYNLFSREFRASL HKGLDSAVEV CVVYGNYSQQ LQVYSKTGFN CDGKLGNESVTFYLQNLYVN QTDIYFCKIE VMYPPPYLDN EKSNGTIIHV KGKHLCPSPLFPGPSKPFWV LVVVGGVLAC YSLLVTVAFI IFWVRSKRSR LLHSDYMNMTPRRPGPTRKH YQPYAPPRDF AAYRSKKVAK KPTNKAPHPK QEPQEINFPDDLPGSNTAAP VQETLHGCQP VTQEDGKESR ISVQERQ 79 CD8a_HuMFMALPVTALLL PLALLLHAAR PQVKLEQSGA EVVKPGASVK LSCKASGFNI E23KDSYMHWLRQ GPGQRLEWIG WIDPENGDTE YAPKFQGKAT FTTDTSANTA _spCD28YLGLSSLRPE DTAVYYCNEG TPTGPYYFDY WGQGTLVTVS SGGGGSGGGG _CD28_SGGGGSENVL TQSPSSMSAS VGDRVNIACS ASSSVSYMHW FQQKPGKSPK CD40LWIYSTSNLA SGVPSRFSGS GSGTDYSLTI SSMQPEDAAT YYCQQRSSYPLTFGGGTKLE IKAAAGSGGS GILVKQSPML VAYDNAVNLS CKYSYNLFSREFRASLHKGL DSAVEVCVVY GNYSQQLQVY SKTGFNCDGK LGNESVTFYLQNLYVNQTDI YFCKIEVMYP PPYLDNEKSN GTIIHVKGKH LCPSPLFPGPSKPFWVLVVV GGVLACYSLL VTVAFIIFWV RSKRSRLLHS DYMNMTPRRPGPTRKHYQPY APPRDFAAYR SKKVAKKPTN KAPHPKQEPQ EINFPDDLPGSNTAAPVQET LHGCQPVTQE DGKESRISVQ ERQ 80 CD2_HuMFEMSFPCKFVAS FLLIFNVSSK GAVSQVKLEQ SGAEWKPGA SVKLSCKASG 23FNIKDSYMHW LRQGPGQRLE WIGWIDPENG DTEYAPKFQG KATFTTDTSA _spCD28NTAYLGLSSL RPEDTAVYYC NEGTPTGPYY FDYWGQGTLV TVSSGGGGSG _CD28_GGGSGGGGSE NVLTQSPSSM SASVGDRVNI ACSASSSVSY MHWFQQKPGK CD40SPKLWIYSTS NLASGVPSRF SGSGSGTDYS LTISSMQPED AATYYCQQRSSYPLTFGGGT KLEIKAAAGS GGSGILVKQS PMLVAYDNAV NLSCKYSYNLFSREFRASLH KGLDSAVEVC VVYGNYSQQL QVYSKTGFNC DGKLGNESVTFYLQNLYVNQ TDIYFCKIEV MYPPPYLDNE KSNGTIIHVK GKHLCPSPLFPGPSKPFWVL VVVGGVLACY SLLVTVAFII FWVRSKRSRL LHSDYMNMTPRRPGPTRKHY QPYAPPRDFA AYRSKKVAKK PTNKAPHPKQ EPQEINFPDDLPGSNTAAPV QETLHGCQPV TQEDGKESRI SVQERQ 81 IL2_HuMFEMYRMQLLSCI ALSLALVTNS QVKLEQSGAE VVKPGASVKL SCKASGFNIK 23DSYMHWLRQG PGQRLEWIGW IDPENGDTEY APKFQGKATF TTDTSANTAY _spCD28LGLSSLRPED TAVYYCNEGT PTGPYYFDYW GQGTLVTVSS GGGGSGGGGS _CD28_GGGGSENVLT QSPSSMSASV GDRVNIACSA SSSVSYMHWF QQKPGKSPKL CD40WIYSTSNLAS GVPSRFSGSG SGTDYSLTIS SMQPEDAATY YCQQRSSYPLTFGGGTKLEI KAAAGSGGSG ILVKQSPMLV AYDNAVNLSC KYSYNLFSREFRASLHKGLD SAVEVCVVYG NYSQQLQVYS KTGFNCDGKL GNESVTFYLQNLYVNQTDIY FCKIEVMYPP PYLDNEKSNG TIIHVKGKHL CPSPLFPGPSKPFWVLVVVG GVLACYSLLV TVAFIIFWVR SKRSRLLHSD YMNMTPRRPGPTRKHYQPYA PPRDFAAYRS KKVAKKPTNK APHPKQEPQE INFPDDLPGSNTAAPVQETL HGCQPVTQED GKESRISVQE RQ 82 GM-MWLQSLLLLG TVACSISQVK LEQSGAEVVK PGASVKLSCK ASGFNIKDSY CSF_HuMFEMHWLRQGPGQ RLEWIGWIDP ENGDTEYAPK FQGKATFTTD TSANTAYLGL 23SSLRPEDTAV YYCNEGTPTG PYYFDYWGQG TLVTVSSGGG GSGGGGSGGG _spCD28GSENVLTQSP SSMSASVGDR VNIACSASSS VSYMHWFQQK PGKSPKLWIY _CD28_STSNLASGVP SRFSGSGSGT DYSLTISSMQ PEDAATYYCQ QRSSYPLTFG CD40GGTKLEIKAA AGSGGSGILV KQSPMLVAYD NAVNLSCKYS YNLFSREFRASLHKGLDSAV EVCVVYGNYS QQLQVYSKTG FNCDGKLGNE SVTFYLQNLYVNQTDIYFCK IEVMYPPPYL DNEKSNGTII HVKGKHLCPS PLFPGPSKPFWVLVVVGGVL ACYSLLVTVA FIIFWVRSKR SRLLHSDYMN MTPRRPGPTRKHYQPYAPPR DFAAYRSKKV AKKPTNKAPH PKQEPQEINF PDDLPGSNTAAPVQETLHGC QPVTQEDGKE SRISVQERQ 83 hIgGk-MEAPAQLLFL LLLWLPDTTR QVKLEQSGAE VVKPGASVKL SCKASGFNIK VIIIDSYMHWLRQG PGQRLEWIGW IDPENGDTEY APKFQGKATF TTDTSANTAY _HuMFE23LGLSSLRPED TAVYYCNEGT PTGPYYFDYW GQGTLVTVSS GGGGSGGGGS _spCD28GGGGSENVLT QSPSSMSASV GDRVNIACSA SSSVSYMHWF QQKPGKSPKL _CD28_WIYSTSNLAS GVPSRFSGSG SGTDYSLTIS SMQPEDAATY YCQQRSSYPL CD40TFGGGTKLEI KAAAGSGGSG ILVKQSPMLV AYDNAVNLSC KYSYNLFSREFRASLHKGLD SAVEVCVVYG NYSQQLQVYS KTGFNCDGKL GNESVTFYLQNLYVNQTDIY FCKIEVMYPP PYLDNEKSNG TIIHVKGKHL CPSPLFPGPSKPFWVLVVVG GVLACYSLLV TVAFIIFWVR SKRSRLLHSD YMNMTPRRPGPTRKHYQPYA PPRDFAAYRS KKVAKKPTNK APHPKQEPQE INFPDDLPGSNTAAPVQETL HGCQPVTQED GKESRISVQE RQ 84 OSM_HuMFEMGVLLTQRTL LSLVLALLFP SMASMQVKLE QSGAEVVKPG ASVKLSCKAS 23GFNIKDSYMH WLRQGPGQRL EWIGWIDPEN GDTEYAPKFQ GKATFTTDTS _spCD8ANTAYLGLSS LRPEDTAVYY CNEGTPTGPY YFDYWGQGTL VTVSSGGGGS _CD28_GGGGSGGGGS ENVLTQSPSS MSASVGDRVN IACSASSSVS YMHWFQQKPG CD40KSPKLWIYST SNLASGVPSR FSGSGSGTDY SLTISSMQPE DAATYYCQQR CTP232SSYPLTFGGG TKLEIKAAAG SGGSGFVPVF LPAKPTTTPA PRPPTPAPTIASQPLSLRPE ACRPAAGGAV HTRGLDFACD IYIWAPLAGT CGVLLLSLVITLYCNHRNRS KRSRLLHSDY MNMTPRRPGP TRKHYQPYAP PRDFAAYRSKKVAKKPTNKA PHPKQEPQEI NFPDDLPGSN TAAPVQETLH GCQPVTQEDG KESRISVQER Q 85CD8a_HuMF MALPVTALLL PLALLLHAAR PQVKLEQSGA EVVKPGASVK LSCKASGFNI E23KDSYMHWLRQ GPGQRLEWIG WIDPENGDTE YAPKFQGKAT FTTDTSANTA _spCD8YLGLSSLRPE DTAVYYCNEG TPTGPYYFDY WGQGTLVTVS SGGGGSGGGG _CD28_SGGGGSENVL TQSPSSMSAS VGDRVNIACS ASSSVSYMHW FQQKPGKSPK CD40LWIYSTSNLA SGVPSRFSGS GSGTDYSLTI SSMQPEDAAT YYCQQRSSYPLTFGGGTKLE IKAAAGSGGS GFVPVFLPAK PTTTPAPRPP TPAPTIASQPLSLRPEACRP AAGGAVHTRG LDFACDIYIW APLAGTCGVL LLSLVITLYCNHRNRSKRSR LLHSDYMNMT PRRPGPTRKH YQPYAPPRDF AAYRSKKVAKKPTNKAPHPK QEPQEINFPD DLPGSNTAAP VQETLHGCQP VTQEDGKESR ISVQERQ 86CD2_HuMFE MSFPCKFVAS FLLIFNVSSK GAVSQVKLEQ SGAEWKPGA SVKLSCKASG 23FNIKDSYMHW LRQGPGQRLE WIGWIDPENG DTEYAPKFQG KATFTTDTSA _spCD8NTAYLGLSSL RPEDTAVYYC NEGTPTGPYY FDYWGQGTLV TVSSGGGGSG _CD28_GGGSGGGGSE NVLTQSPSSM SASVGDRVNI ACSASSSVSY MHWFQQKPGK CD40SPKLWIYSTS NLASGVPSRF SGSGSGTDYS LTISSMQPED AATYYCQQRSSYPLTFGGGT KLEIKAAAGS GGSGFVPVFL PAKPTTTPAP RPPTPAPTIASQPLSLRPEA CRPAAGGAVH TRGLDFACDI YIWAPLAGTC GVLLLSLVITLYCNHRNRSK RSRLLHSDYM NMTPRRPGPT RKHYQPYAPP RDFAAYRSKKVAKKPTNKAP HPKQEPQEIN FPDDLPGSNT AAPVQETLHG CQPVTQEDGK ESRISVQERQ 87IL2_HuMFE MYRMQLLSCI ALSLALVTNS QVKLEQSGAE VVKPGASVKL SCKASGFNIK 23DSYMHWLRQG PGQRLEWIGW IDPENGDTEY APKFQGKATF TTDTSANTAY _spCD8LGLSSLRPED TAVYYCNEGT PTGPYYFDYW GQGTLVTVSS GGGGSGGGGS _CD28_GGGGSENVLT QSPSSMSASV GDRVNIACSA SSSVSYMHWF QQKPGKSPKL CD40WIYSTSNLAS GVPSRFSGSG SGTDYSLTIS SMQPEDAATY YCQQRSSYPLTFGGGTKLEI KAAAGSGGSG FVPVFLPAKP TTTPAPRPPT PAPTIASQPLSLRPEACRPA AGGAVHTRGL DFACDIYIWA PLAGTCGVLL LSLVITLYCNHRNRSKRSRL LHSDYMNMTP RRPGPTRKHY QPYAPPRDFA AYRSKKVAKKPTNKAPHPKQ EPQEINFPDD LPGSNTAAPV QETLHGCQPV TQEDGKESRI SVQERQ 88 GM-MWLQSLLLLG TVACSISQVK LEQSGAEVVK PGASVKLSCK ASGFNIKDSY CSF_HuMFEMHWLRQGPGQ RLEWIGWIDP ENGDTEYAPK FQGKATFTTD TSANTAYLGL 23SSLRPEDTAV YYCNEGTPTG PYYFDYWGQG TLVTVSSGGG GSGGGGSGGG _spCD8GSENVLTQSP SSMSASVGDR VNIACSASSS VSYMHWFQQK PGKSPKLWIY _CD28_STSNLASGVP SRFSGSGSGT DYSLTISSMQ PEDAATYYCQ QRSSYPLTFG CD40GGTKLEIKAA AGSGGSGFVP VFLPAKPTTT PAPRPPTPAP TIASQPLSLRPEACRPAAGG AVHTRGLDFA CDIYIWAPLA GTCGVLLLSL VITLYCNHRNRSKRSRLLHS DYMNMTPRRP GPTRKHYQPY APPRDFAAYR SKKVAKKPTNKAPHPKQEPQ EINFPDDLPG SNTAAPVQET LHGCQPVTQE DGKESRISVQ ERQ 89 hIgGk-MEAPAQLLFL LLLWLPDTTR QVKLEQSGAE VVKPGASVKL SCKASGFNIK VIIIDSYMHWLRQG PGQRLEWIGW IDPENGDTEY APKFQGKATF TTDTSANTAY _HuMFE23LGLSSLRPED TAVYYCNEGT PTGPYYFDYW GQGTLVTVSS GGGGSGGGGS _spCD8GGGGSENVLT QSPSSMSASV GDRVNIACSA SSSVSYMHWF QQKPGKSPKL _CD28_WIYSTSNLAS GVPSRFSGSG SGTDYSLTIS SMQPEDAATY YCQQRSSYPL CD40TFGGGTKLEI KAAAGSGGSG FVPVFLPAKP TTTPAPRPPT PAPTIASQPLSLRPEACRPA AGGAVHTRGL DFACDIYIWA PLAGTCGVLL LSLVITLYCNHRNRSKRSRL LHSDYMNMTP RRPGPTRKHY QPYAPPRDFA AYRSKKVAKKPTNKAPHPKQ EPQEINFPDD LPGSNTAAPV QETLHGCQPV TQEDGKESRI SVQERQ 90OSM_HuMFE MGVLLTQRTL LSLVLALLFP SMASMQVKLE QSGAEVVKPG ASVKLSCKAS 23GFNIKDSYMH WLRQGPGQRL EWIGWIDPEN GDTEYAPKFQ GKATFTTDTS _CD28TMANTAYLGLSS LRPEDTAVYY CNEGTPTGPY YFDYWGQGTL VTVSSGGGGS _CD28_GGGGSGGGGS ENVLTQSPSS MSASVGDRVN IACSASSSVS YMHWFQQKPG CD40KSPKLWIYST SNLASGVPSR FSGSGSGTDY SLTISSMQPE DAATYYCQQRSSYPLTFGGG TKLEIKAAAG SGGSGFWVLV VVGGVLACYS LLVTVAFIIFWVRSKRSRLL HSDYMNMTPR RPGPTRKHYQ PYAPPRDFAA YRSKKVAKKPTNKAPHPKQE PQEINFPDDL PGSNTAAPVQ ETLHGCQPVT QEDGKESRIS VQERQ 91CD8a_HuMF MALPVTALLL PLALLLHAAR PQVKLEQSGA EVVKPGASVK LSCKASGFNI E23KDSYMHWLRQ GPGQRLEWIG WIDPENGDTE YAPKFQGKAT FTTDTSANTA _CD28TMYLGLSSLRPE DTAVYYCNEG TPTGPYYFDY WGQGTLVTVS SGGGGSGGGG _CD28_SGGGGSENVL TQSPSSMSAS VGDRVNIACS ASSSVSYMHW FQQKPGKSPK CD40LWIYSTSNLA SGVPSRFSGS GSGTDYSLTI SSMQPEDAAT YYCQQRSSYPLTFGGGTKLE IKAAAGSGGS GFWVLVVVGG VLACYSLLVT VAFIIFWVRSKRSRLLHSDY MNMTPRRPGP TRKHYQPYAP PRDFAAYRSK KVAKKPTNKAPHPKQEPQEI NFPDDLPGSN TAAPVQETLH GCQPVTQEDG KESRISVQER Q 92 CD2_HuMFEMSFPCKFVAS FLLIFNVSSK GAVSQVKLEQ SGAEVVKPGA SVKLSCKASG 23FNIKDSYMHW LRQGPGQRLE WIGWIDPENG DTEYAPKFQG KATFTTDTSA _CD28TMNTAYLGLSSL RPEDTAVYYC NEGTPTGPYY FDYWGQGTLV TVSSGGGGSG _CD28_GGGSGGGGSE NVLTQSPSSM SASVGDRVNI ACSASSSVSY MHWFQQKPGK CD40SPKLWIYSTS NLASGVPSRF SGSGSGTDYS LTISSMQPED AATYYCQQRSSYPLTFGGGT KLEIKAAAGS GGSGFWVLVV VGGVLACYSL LVTVAFIIFWVRSKRSRLLH SDYMNMTPRR PGPTRKHYQP YAPPRDFAAY RSKKVAKKPTNKAPHPKQEP QEINFPDDLP GSNTAAPVQE TLHGCQPVTQ EDGKESRISV QERQ 93 IL2_HuMFEMYRMQLLSCI ALSLALVTNS QVKLEQSGAE VVKPGASVKL SCKASGFNIK 23DSYMHWLRQG PGQRLEWIGW IDPENGDTEY APKFQGKATF TTDTSANTAY _CD28TMLGLSSLRPED TAVYYCNEGT PTGPYYFDYW GQGTLVTVSS GGGGSGGGGS _CD28_GGGGSENVLT QSPSSMSASV GDRVNIACSA SSSVSYMHWF QQKPGKSPKL CD40WIYSTSNLAS GVPSRFSGSG SGTDYSLTIS SMQPEDAATY YCQQRSSYPLTFGGGTKLEI KAAAGSGGSG FWVLVVVGGV LACYSLLVTV AFIIFWVRSKRSRLLHSDYM NMTPRRPGPT RKHYQPYAPP RDFAAYRSKK VAKKPTNKAPHPKQEPQEIN FPDDLPGSNT AAPVQETLHG CQPVTQEDGK ESRISVQERQ 94 GM-MWLQSLLLLG TVACSISQVK LEQSGAEVVK PGASVKLSCK ASGFNIKDSY CSF_HuMFEMHWLRQGPGQ RLEWIGWIDP ENGDTEYAPK FQGKATFTTD TSANTAYLGL 23SSLRPEDTAV YYCNEGTPTG PYYFDYWGQG TLVTVSSGGG GSGGGGSGGG _CD28TMGSENVLTQSP SSMSASVGDR VNIACSASSS VSYMHWFQQK PGKSPKLWIY _CD28_STSNLASGVP SRFSGSGSGT DYSLTISSMQ PEDAATYYCQ QRSSYPLTFG CD40GGTKLEIKAA AGSGGSGFWV LVVVGGVLAC YSLLVTVAFI IFWVRSKRSRLLHSDYMNMT PRRPGPTRKH YQPYAPPRDF AAYRSKKVAK KPTNKAPHPKQEPQEINFPD DLPGSNTAAP VQETLHGCQP VTQEDGKESR ISVQERQ 95 hIgGk-MEAPAQLLFL LLLWLPDTTR QVKLEQSGAE VVKPGASVKL SCKASGFNIK VIIIDSYMHWLRQG PGQRLEWIGW IDPENGDTEY APKFQGKATF TTDTSANTAY _HuMFE23LGLSSLRPED TAVYYCNEGT PTGPYYFDYW GQGTLVTVSS GGGGSGGGGS _CD28TMGGGGSENVLT QSPSSMSASV GDRVNIACSA SSSVSYMHWF QQKPGKSPKL _CD28_WIYSTSNLAS GVPSRFSGSG SGTDYSLTIS SMQPEDAATY YCQQRSSYPL CD40TFGGGTKLEI KAAAGSGGSG FWVLVVVGGV LACYSLLVTV AFIIFWVRSKRSRLLHSDYM NMTPRRPGPT RKHYQPYAPP RDFAAYRSKK VAKKPTNKAPHPKQEPQEIN FPDDLPGSNT AAPVQETLHG CQPVTQEDGK ESRISVQERQ 96 OSM_CEA6MGVLLTQRTL LSLVLALLFP SMASMQVQLV QSGAEVKKPG SSVKVSCKAS _spCD28GGTFSNSPIN WLRQAPGQGL EWMGSIIPSF GTANYAQKFQ GRLTITADES _CD28_TSTAYMELSS LRSEDTAVYY CAGRSHNYEL YYYYMDVWGQ GTMVTVSSGG CD40GGSGGGGSGG GGSDIQMTQS PSTLSASIGD RVTITCRASE GIYHWLAWYQ CTP221QKPGKAPKLL IYKASSLASG APSRFSGSGS GTDFTLTISS LQPDDFATYYCQQYSNYPLT FGGGTKLEIK RAAAGSGGSG ILVKQSPMLV AYDNAVNLSCKYSYNLFSRE FRASLHKGLD SAVEVCVVYG NYSQQLQVYS KTGFNCDGKLGNESVTFYLQ NLYVNQTDIY FCKIEVMYPP PYLDNEKSNG TIIHVKGKHLCPSPLFPGPS KPFWVLVVVG GVLACYSLLV TVAFIIFWVR SKRSRLLHSDYMNMTPRRPG PTRKHYQPYA PPRDFAAYRS KKVAKKPTNK APHPKQEPQEINFPDDLPGS NTAAPVQETL HGCQPVTQED GKESRISVQE RQ 97 CD8a_CEA6MALPVTALLL PLALLLHAAR PQVQLVQSGA EVKKPGSSVK VSCKASGGTF _spCD28SNSPINWLRQ APGQGLEWMG SIIPSFGTAN YAQKFQGRLT ITADESTSTA _CD28_YMELSSLRSE DTAVYYCAGR SHNYELYYYY MDVWGQGTMV TVSSGGGGSG CD40GGGSGGGGSD IQMTQSPSTL SASIGDRVTI TGRASEGIYH WLAWYQQKPGKAPKLLIYKA SSLASGAPSR FSGSGSGTDF TLTISSLQPD DFATYYCQQYSNYPLTFGGG TKLEIKRAAA GSGGSGILVK QSPMLVAYDN AVNLSCKYSYNLFSREFRAS LHKGLDSAVE VCVVYGNYSQ QLQVYSKTGF NCDGKLGNESVTFYLQNLYV NQTDIYFCKI EVMYPPPYLD NEKSNGTIIH VKGKHLCPSPLFPGPSKPFW VLVVVGGVLA CYSLLVTVAF IIFWVRSKRS RLLHSDYMNMTPRRPGPTRK HYQPYAPPRD FAAYRSKKVA KKPTNKAPHP KQEPQEINFPDDLPGSNTAA PVQETLHGCQ PVTQEDGKES RISVQERQ 98 CD2_CEA6MSFPCKFVAS FLLIFNVSSK GAVSQVQLVQ SGAEVKKPGS SVKVSCKASG _spCD28GTFSNSPINW LRQAPGQGLE WMGSIIPSFG TANYAQKFQG RLTITADEST _CD28_STAYMELSSL RSEDTAVYYC AGRSHNYELY YYYMDVWGQG TMVTVSSGGG CD40GSGGGGSGGG GSDIQMTQSP STLSASIGDR VTITCRASEG IYHWLAWYQQKPGKAPKLLI YKASSLASGA PSRFSGSGSG TDFTLTISSL QPDDFATYYCQQYSNYPLTF GGGTKLEIKR AAAGSGGSGI LVKQSPMLVA YDNAVNLSCKYSYNLFSREF RASLHKGLDS AVEVCVVYGN YSQQLQVYSK TGFNCDGKLGNESVTFYLQN LYVNQTDIYF CKIEVMYPPP YLDNEKSNGT IIHVKGKHLCPSPLFPGPSK PFWVLVVVGG VLACYSLLVT VAFIIFWVRS KRSRLLHSDYMNMTPRRPGP TRKHYQPYAP PRDFAAYRSK KVAKKPTNKA PHPKQEPQEINFPDDLPGSN TAAPVQETLH GCQPVTQEDG KESRISVQER Q 99 IL2_CEA6MYRMQLLSCI ALSLALVTNS QVQLVQSGAE VKKPGSSVKV SCKASGGTFS _spCD28NSPINWLRQA PGQGLEWMGS IIPSFGTANY AQKFQGRLTI TADESTSTAY _CD28_MELSSLRSED TAVYYCAGRS HNYELYYYYM DVWGQGTMVT VSSGGGGSGG CD40GGSGGGGSDI QMTQSPSTLS ASIGDRVTIT CRASEGIYHW LAWYQQKPGKAPKLLIYKAS SLASGAPSRF SGSGSGTDFT LTISSLQPDD FATYYCQQYSNYPLTFGGGT KLEIKRAAAG SGGSGILVKQ SPMLVAYDNA VNLSCKYSYNLFSREFRASL HKGLDSAVEV CVVYGNYSQQ LQVYSKTGFN CDGKLGNESVTFYLQNLYVN QTDIYFCKIE VMYPPPYLDN EKSNGTIIHV KGKHLCPSPLFPGPSKPFWV LVVVGGVLAC YSLLVTVAFI IFWVRSKRSR LLHSDYMNMTPRRPGPTRKH YQPYAPPRDF AAYRSKKVAK KPTNKAPHPK QEPQEINFPDDLPGSNTAAP VQETLHGCQP VTQEDGKESR ISVQERQ 100 GM-MWLQSLLLLG TVACSISQVQ LVQSGAEVKK PGSSVKVSCK ASGGTFSNSP CSF_CEA6INWLRQAPGQ GLEWMGSIIP SFGTANYAQK FQGRLTITAD ESTSTAYMEL _spCD28SSLRSEDTAV YYCAGRSHNY ELYYYYMDVW GQGTMVTVSS GGGGSGGGGS _CD28_GGGGSDIQMT QSPSTLSASI GDRVTITCRA SEGIYHWLAW YQQKPGKAPK CD40LLIYKASSLA SGAPSRFSGS GSGTDFTLTI SSLQPDDFAT YYCQQYSNYPLTFGGGTKLE IKRAAAGSGG SGILVKQSPM LVAYDNAVNL SCKYSYNLFSREFRASLHKG LDSAVEVCVV YGNYSQQLQV YSKTGFNCDG KLGNESVTFYLQNLYVNQTD IYFCKIEVMY PPPYLDNEKS NGTIIHVKGK HLCPSPLFPGPSKPFWVLVV VGGVLACYSL LVTVAFIIFW VRSKRSRLLH SDYMNMTPRRPGPTRKHYQP YAPPRDFAAY RSKKVAKKPT NKAPHPKQEP QEINFPDDLPGSNTAAPVQE TLHGCQPVTQ EDGKESRISV QERQ 101 hIgGk-MEAPAQLLFL LLLWLPDTTR QVQLVQSGAE VKKPGSSVKV SCKASGGTFS VIII_CEA6NSPINWLRQA PGQGLEWMGS IIPSFGTANY AQKFQGRLTI TADESTSTAY _spCD28MELSSLRSED TAVYYCAGRS HNYELYYYYM DVWGQGTMVT VSSGGGGSGG _CD28_GGSGGGGSDI QMTQSPSTLS ASIGDRVTIT CRASEGIYHW LAWYQQKPGK CD40APKLLIYKAS SLASGAPSRF SGSGSGTDFT LTISSLQPDD FATYYCQQYSNYPLTFGGGT KLEIKRAAAG SGGSGILVKQ SPMLVAYDNA VNLSCKYSYNLFSREFRASL HKGLDSAVEV CVVYGNYSQQ LQVYSKTGFN CDGKLGNESVTFYLQNLYVN QTDIYFCKIE VMYPPPYLDN EKSNGTIIHV KGKHLCPSPLFPGPSKPFWV LVVVGGVLAC YSLLVTVAFI IFWVRSKRSR LLHSDYMNMTPRRPGPTRKH YQPYAPPRDF AAYRSKKVAK KPTNKAPHPK QEPQEINFPDDLPGSNTAAP VQETLHGCQP VTQEDGKESR ISVQERQ 102 OSM_CEA6MGVLLTQRTL LSLVLALLFP SMASMQVQLV QSGAEVKKPG SSVKVSCKAS _spCD8GGTFSNSPIN WLRQAPGQGL EWMGSIIPSF GTANYAQKFQ GRLTITADES _CD28_TSTAYMELSS LRSEDTAVYY CAGRSHNYEL YYYYMDVWGQ GTMVTVSSGG CD40GGSGGGGSGG GGSDIQMTQS PSTLSASIGD RVTITCRASE GIYHWLAWYQQKPGKAPKLL IYKASSLASG APSRFSGSGS GTDFTLTISS LQPDDFATYYCQQYSNYPLT FGGGTKLEIK RAAAGSGGSG FVPVFLPAKP TTTPAPRPPTPAPTIASQPL SLRPEACRPA AGGAVHTRGL DFACDIYIWA PLAGTCGVLLLSLVITLYCN HRNRSKRSRL LHSDYMNMTP RRPGPTRKHY QPYAPPRDFAAYRSKKVAKK PTNKAPHPKQ EPQEINFPDD LPGSNTAAPV QETLHGCQPV TQEDGKESRI SVQERQ103 CD8a_CEA6 MALPVTALLL PLALLLHAAR PQVQLVQSGA EVKKPGSSVK VSCKASGGTF_spCD8 SNSPINWLRQ APGQGLEWMG SIIPSFGTAN YAQKFQGRLT ITADESTSTA _CD28_YMELSSLRSE DTAVYYCAGR SHNYELYYYY MDVWGQGTMV TVSSGGGGSG CD40GGGSGGGGSD IQMTQSPSTL SASIGDRVTI TCRASEGIYH WLAWYQQKPGKAPKLLIYKA SSLASGAPSR FSGSGSGTDF TLTISSLQPD DFATYYCQQYSNYPLTFGGG TKLEIKRAAA GSGGSGFVPV FLPAKPTTTP APRPPTPAPTIASQPLSLRP EACRPAAGGA VHTRGLDFAC DIYIWAPLAG TCGVLLLSLVITLYCNHRNR SKRSRLLHSD YMNMTPRRPG PTRKHYQPYA PPRDFAAYRSKKVAKKPTNK APHPKQEPQE INFPDDLPGS NTAAPVQETL HGCQPVTQED GKESRISVQE RQ 104CD2_CEA6 MSFPCKFVAS FLLIFNVSSK GAVSQVQLVQ SGAEVKKPGS SVKVSCKASG _spCD8GTFSNSPINW LRQAPGQGLE WMGSIIPSFG TANYAQKFQG RLTITADEST _CD28_STAYMELSSL RSEDTAVYYC AGRSHNYELY YYYMDVWGQG TMVTVSSGGG CD40GSGGGGSGGG GSDIQMTQSP STLSASIGDR VTITCRASEG IYHWLAWYQQKPGKAPKLLI YKASSLASGA PSRFSGSGSG TDFTLTISSL QPDDFATYYCQQYSNYPLTF GGGTKLEIKR AAAGSGGSGF VPVFLPAKPT TTPAPRPPTPAPTIASQPLS LRPEACRPAA GGAVHTRGLD FACDIYIWAP LAGTCGVLLLSLVITLYCNH RNRSKRSRLL HSDYMNMTPR RPGPTRKHYQ PYAPPRDFAAYRSKKVAKKP TNKAPHPKQE PQEINFPDDL PGSNTAAPVQ ETLHGCQPVT QEDGKESRIS VQERQ105 IL2_CEA6 MYRMQLLSCI ALSLALVTNS QVQLVQSGAE VKKPGSSVKV SCKASGGTFS_spCD8 NSPINWLRQA PGQGLEWMGS IIPSEGTANY AQKFQGRLTI TADESTSTAY _CD28_MELSSLRSED TAVYYCAGRS HNYELYYYYM DVWGQGTMVT VSSGGGGSGG CD40GGSGGGGSDI QMTQSPSTLS ASIGDRVTIT CRASEGIYHW LAWYQQKPGKAPKLLIYKAS SLASGAPSRF SGSGSGTDFT LTISSLQPDD FATYYCQQYSNYPLTFGGGT KLEIKRAAAG SGGSGFVPVF LPAKPTTTPA PRPPTPAPTIASQPLSLRPE ACRPAAGGAV HTRGLDFACD IYIWAPLAGT CGVLLLSLVITLYCNHRNRS KRSRLLHSDY MNMTPRRPGP TRKHYQPYAP PRDFAAYRSKKVAKKPTNKA PHPKQEPQEI NFPDDLPGSN TAAPVQETLH GCQPVTQEDG KESRISVQER Q 106GM- MWLQSLLLLG TVACSISQVQ LVQSGAEVKK PGSSVKVSCK ASGGTFSNSP CSF_CEA6INWLRQAPGQ GLEWMGSIIP SFGTANYAQK FQGRLTITAD ESTSTAYMEL _spCD8SSLRSEDTAV YYCAGRSHNY ELYYYYMDVW GQGTMVTVSS GGGGSGGGGS _CD28_GGGGSDIQMT QSPSTLSASI GDRVTITCRA SEGIYHWLAW YQQKPGKAPK CD40LLIYKASSLA SGAPSRFSGS GSGTDFTLTI SSLQPDDFAT YYCQQYSNYPLTFGGGTKLE IKRAAAGSGG SGFVPVFLPA KPTTTPAPRP PTPAPTIASQPLSLRPEACR PAAGGAVHTR GLDFACDIYI WAPLAGTCGV LLLSLVITLYCNHRNRSKRS RLLHSDYMNM TPRRPGPTRK HYQPYAPPRD FAAYRSKKVAKKPTNKAPHP KQEPQEINFP DDLPGSNTAA PVQETLHGCQ PVTQEDGKES RISVQERQ 107hIgGk- MEAPAQLLFL LLLWLPDTTR QVQLVQSGAE VKKPGSSVKV SCKASGGTFS VIIINSPINWLRQA PGQGLEWMGS IIPSEGTANY AQKFQGRLTI TADESTSTAY _CEA6MELSSLRSED TAVYYCAGRS HNYELYYYYM DVWGQGTMVT VSSGGGGSGG _spCD8GGSGGGGSDI QMTQSPSTLS ASIGDRVTIT CRASEGIYHW LAWYQQKPGK _CD28_APKLLIYKAS SLASGAPSRF SGSGSGTDFT LTISSLQPDD FATYYCQQYS CD40NYPLTFGGGT KLEIKRAAAG SGGSGFVPVF LPAKPTTTPA PRPPTPAPTIASQPLSLRPE ACRPAAGGAV HTRGLDFACD IYIWAPLAGT CGVLLLSLVITLYCNHRNRS KRSRLLHSDY MNMTPRRPGP TRKHYQPYAP PRDFAAYRSKKVAKKPTNKA PHPKQEPQEI NFPDDLPGSN TAAPVQETLH GCQPVTQEDG KESRISVQER Q 108OSM_CEA6 MGVLLTQRTL LSLVLALLFP SMASMQVQLV QSGAEVKKPG SSVKVSCKAS _CD28TMGGTFSNSPIN WLRQAPGQGL EWMGSIIPSF GTANYAQKFQ GRLTITADES _CD28_TSTAYMELSS LRSEDTAVYY CAGRSHNYEL YYYYMDVWGQ GTMVTVSSGG CD40GGSGGGGSGG GGSDIQMTQS PSTLSASIGD RVTITCRASE GIYHWLAWYQQKPGKAPKLL IYKASSLASG APSRFSGSGS GTDFTLTISS LQPDDFATYYCQQYSNYPLT FGGGTKLEIK RAAAGSGGSG FWVLVVVGGV LACYSLLVTVAFIIFWVRSK RSRLLHSDYM NMTPRRPGPT RKHYQPYAPP RDFAAYRSKKVAKKPTNKAP HPKQEPQEIN FPDDLPGSNT AAPVQETLHG CQPVTQEDGK ESRISVQERQ 109CD8a_CEA6 MALPVTALLL PLALLLHAAR PQVQLVQSGA EVKKPGSSVK VSCKASGGTF _CD28TMSNSPINWLRQ APGQGLEWMG SIIPSFGTAN YAQKFQGRLT ITADESTSTA _CD28_YMELSSLRSE DTAVYYCAGR SHNYELYYYY MDVWGQGTMV TVSSGGGGSG CD40GGGSGGGGSD IQMTQSPSTL SASIGDRVTI TCRASEGIYH WLAWYQQKPGKAPKLLIYKA SSLASGAPSR FSGSGSGTDF TLTISSLQPD DFATYYCQQYSNYPLTFGGG TKLEIKRAAA GSGGSGFWVL VVVGGVLACY SLLVTVAFIIFWVRSKRSRL LHSDYMNMTP RRPGPTRKHY QPYAPPRDFA AYRSKKVAKKPTNKAPHPKQ EPQEINFPDD LPGSNTAAPV QETLHGCQPV TQEDGKESRI SVQERQ 110CD2_CEA6 MSFPCKFVAS FLLIFNVSSK GAVSQVQLVQ SGAEVKKPGS SVKVSCKASG _CD28TMGTFSNSPINW LRQAPGQGLE WMGSIIPSFG TANYAQKFQG RLTITADEST _CD28_STAYMELSSL RSEDTAVYYC AGRSHNYELY YYYMDVWGQG TMVTVSSGGG CD40GSGGGGSGGG GSDIQMTQSP STLSASIGDR VTITCRASEG IYHWLAWYQQKPGKAPKLLI YKASSLASGA PSRFSGSGSG TDFTLTISSL QPDDFATYYCQQYSNYPLTF GGGTKLEIKR AAAGSGGSGF WVLVVVGGVL ACYSLLVTVAFIIFWVRSKR SRLLHSDYMN MTPRRPGPTR KHYQPYAPPR DFAAYRSKKVAKKPTNKAPH PKQEPQEINF PDDLPGSNTA APVQETLHGC QPVTQEDGKE SRISVQERQ 111IL2_CEA6 MYRMQLLSCI ALSLALVTNS QVQLVQSGAE VKKPGSSVKV SCKASGGTFS _CD28TMNSPINWLRQA PGQGLEWMGS IIPSEGTANY AQKFQGRLTI TADESTSTAY _CD28_MELSSLRSED TAVYYCAGRS HNYELYYYYM DVWGQGTMVT VSSGGGGSGG CD40GGSGGGGSDI QMTQSPSTLS ASIGDRVTIT CRASEGIYHW LAWYQQKPGKAPKLLIYKAS SLASGAPSRF SGSGSGTDFT LTISSLQPDD FATYYCQQYSNYPLTFGGGT KLEIKRAAAG SGGSGFWVLV VVGGVLACYS LLVTVAFIIFWVRSKRSRLL HSDYMNMTPR RPGPTRKHYQ PYAPPRDFAA YRSKKVAKKPTNKAPHPKQE PQEINFPDDL PGSNTAAPVQ ETLHGCQPVT QEDGKESRIS VQERQ 112 GM-MWLQSLLLLG TVACSISQVQ LVQSGAEVKK PGSSVKVSCK ASGGTFSNSP CSF_CEA6INWLRQAPGQ GLEWMGSIIP SFGTANYAQK FQGRLTITAD ESTSTAYMEL _CD28TMSSLRSEDTAV YYCAGRSHNY ELYYYYMDVW GQGTMVTVSS GGGGSGGGGS _CD28_GGGGSDIQMT QSPSTLSASI GDRVTITCRA SEGIYHWLAW YQQKPGKAPK CD40LLIYKASSLA SGAPSRFSGS GSGTDFTLTI SSLQPDDFAT YYCQQYSNYPLTFGGGTKLE IKRAAAGSGG SGFWVLVVVG GVLACYSLLV TVAFIIFWVRSKRSRLLHSD YMNMTPRRPG PTRKHYQPYA PPRDFAAYRS KKVAKKPTNKAPHPKQEPQE INFPDDLPGS NTAAPVQETL HGCQPVTQED GKESRISVQE RQ 113 hIgGk-MEAPAQLLFL LLLWLPDTTR QVQLVQSGAE VKKPGSSVKV SCKASGGTFS VIIINSPINWLRQA PGQGLEWMGS IIPSEGTANY AQKFQGRLTI TADESTSTAY _CEA6MELSSLRSED TAVYYCAGRS HNYELYYYYM DVWGQGTMVT VSSGGGGSGG _CD28TMGGSGGGGSDI QMTQSPSTLS ASIGDRVTIT CRASEGIYHW LAWYQQKPGK _CD28_APKLLIYKAS SLASGAPSRF SGSGSGTDFT LTISSLQPDD FATYYCQQYS CD40NYPLTFGGGT KLEIKRAAAG SGGSGFWVLV VVGGVLACYS LLVTVAFIIFWVRSKRSRLL HSDYMNMTPR RPGPTRKHYQ PYAPPRDFAA YRSKKVAKKPTNKAPHPKQE PQEINFPDDL PGSNTAAPVQ ETLHGCQPVT QEDGKESRIS VQERQ 114OSM_BW431 MGVLLTQRTL LSLVLALLFP SMASMQLQES GPGLVRPSQT LSLTCTVSGF /26TISSGYSWHW VRQPPGRGLE WIGYIQYSGI TNYNPSLKSR VTMLVDTSKN _spCD28QFSLRLSSVT AADTAVYYCA REDYDYHWYF DVWGQGSLVT VSSGGGGSGG _CD28_GGSGGGGSGV HSDIQMTQSP SSLSASVGDR VTITCSTSSS VSYMHWYQQK CD40PGKAPKLLIY STSNLASGVP SRFSGSGSGT DFTFTISSLQ PEDIATYYCH CTP222QWSSYPTFGQ GTKVEIKRAA AGSGGSGILV KQSPMLVAYD NAVNLSCKYSYNLFSREFRA SLHKGLDSAV EVCVVYGNYS QQLQVYSKTG FNCDGKLGNESVTFYLQNLY VNQTDIYFCK IEVMYPPPYL DNEKSNGTII HVKGKHLCPSPLFPGPSKPF WVLVVVGGVL ACYSLLVTVA FIIFWVRSKR SRLLHSDYMNMTPRRPGPTR KHYQPYAPPR DFAAYRSKKV AKKPTNKAPH PKQEPQEINFPDDLPGSNTA APVQETLHGC QPVTQEDGKE SRISVQERQ 115 CD8a_BW43MALPVTALLL PLALLLHAAR PQLQESGPGL VRPSQTLSLT CTVSGFTISS 1/26GYSWHWVRQP PGRGLEWIGY IQYSGITNYN PSLKSRVTML VDTSKNQFSL _spCD28RLSSVTAADT AVYYCAREDY DYHWYFDVWG QGSLVTVSSG GGGSGGGGSG _CD28_GGGSGVHSDI QMTQSPSSLS ASVGDRVTIT CSTSSSVSYM HWYQQKPGKA CD40PKLLIYSTSN LASGVPSRFS GSGSGTDFTF TISSLQPEDI ATYYCHQWSSYPTFGQGTKV EIKRAAAGSG GSGILVKQSP MLVAYDNAVN LSCKYSYNLFSREFRASLHK GLDSAVEVCV VYGNYSQQLQ VYSKTGFNCD GKLGNESVTFYLQNLYVNQT DIYFCKIEVM YPPPYLDNEK SNGTIIHVKG KHLCPSPLFPGPSKPFWVLV VVGGVLACYS LLVTVAFIIF WVRSKRSRLL HSDYMNMTPRRPGPTRKHYQ PYAPPRDFAA YRSKKVAKKP TNKAPHPKQE PQEINFPDDLPGSNTAAPVQ ETLHGCQPVT QEDGKESRIS VQERQ 116 CD2_BW431MSFPCKFVAS FLLIFNVSSK GAVSQLQESG PGLVRPSQTL SLTCTVSGFT /26ISSGYSWHWV RQPPGRGLEW IGYIQYSGIT NYNPSLKSRV TMLVDTSKNQ _spCD28FSLRLSSVTA ADTAVYYCAR EDYDYHWYFD VWGQGSLVTV SSGGGGSGGG _CD28_GSGGGGSGVH SDIQMTQSPS SLSASVGDRV TITCSTSSSV SYMHWYQQKP CD40GKAPKLLIYS TSNLASGVPS RFSGSGSGTD FTFTISSLQP EDIATYYCHQWSSYPTFGQG TKVEIKRAAA GSGGSGILVK QSPMLVAYDN AVNLSCKYSYNLFSREFRAS LHKGLDSAVE VCVVYGNYSQ QLQVYSKTGF NCDGKLGNESVTFYLQNLYV NQTDIYFCKI EVMYPPPYLD NEKSNGTIIH VKGKHLCPSPLFPGPSKPFW VLVVVGGVLA CYSLLVTVAF IIFWVRSKRS RLLHSDYMNMTPRRPGPTRK HYQPYAPPRD FAAYRSKKVA KKPTNKAPHP KQEPQEINFPDDLPGSNTAA PVQETLHGCQ PVTQEDGKES RISVQERQ 117 IL2_BW431MYRMQLLSCI ALSLALVTNS QLQESGPGLV RPSQTLSLTC TVSGFTISSG /26YSWHWVRQPP GRGLEWIGYI QYSGITNYNP SLKSRVTMLV DTSKNQFSLR _spCD28LSSVTAADTA VYYCAREDYD YHWYFDVWGQ GSLVTVSSGG GGSGGGGSGG _CD28_GGSGVHSDIQ MTQSPSSLSA SVGDRVTITC STSSSVSYMH WYQQKPGKAP CD40KLLIYSTSNL ASGVPSRFSG SGSGTDFTFT ISSLQPEDIA TYYCHQWSSYPTFGQGTKVE IKRAAAGSGG SGILVKQSPM LVAYDNAVNL SCKYSYNLFSREFRASLHKG LDSAVEVCVV YGNYSQQLQV YSKTGFNCDG KLGNESVTFYLQNLYVNQTD IYFCKIEVMY PPPYLDNEKS NGTIIHVKGK HLCPSPLFPGPSKPFWVLVV VGGVLACYSL LVTVAFIIFW VRSKRSRLLH SDYMNMTPRRPGPTRKHYQP YAPPRDFAAY RSKKVAKKPT NKAPHPKQEP QEINFPDDLPGSNTAAPVQE TLHGCQPVTQ EDGKESRISV QERQ 118 GM-CSFMWLQSLLLLG TVACSISQLQ ESGPGLVRPS QTLSLTCTVS GFTISSGYSW _BW431/26HWVRQPPGRG LEWIGYIQYS GITNYNPSLK SRVTMLVDTS KNQFSLRLSS _spCD28VTAADTAVYY CAREDYDYHW YFDVWGQGSL VTVSSGGGGS GGGGSGGGGS _CD28_GVHSDIQMTQ SPSSLSASVG DRVTITCSTS SSVSYMHWYQ QKPGKAPKLL CD40IYSTSNLASG VPSRFSGSGS GTDFTFTISS LQPEDIATYY CHQWSSYPTFGQGTKVEIKR AAAGSGGSGI LVKQSPMLVA YDNAVNLSCK YSYNLFSREFRASLHKGLDS AVEVCVVYGN YSQQLQVYSK TGFNCDGKLG NESVTFYLQNLYVNQTDIYF CKIEVMYPPP YLDNEKSNGT IIHVKGKHLC PSPLFPGPSKPFWVLVVVGG VLACYSLLVT VAFIIFWVRS KRSRLLHSDY MNMTPRRPGPTRKHYQPYAP PRDFAAYRSK KVAKKPTNKA PHPKQEPQEI NFPDDLPGSNTAAPVQETLH GCQPVTQEDG KESRISVQER Q 119 hIgGk-MEAPAQLLFL LLLWLPDTTR QLQESGPGLV RPSQTLSLTC TVSGFTISSG VIIIYSWHWVRQPP GRGLEWIGYI QYSGITNYNP SLKSRVTMLV DTSKNQFSLR _BW431/26LSSVTAADTA VYYCAREDYD YHWYFDVWGQ GSLVTVSSGG GGSGGGGSGG _spCD28GGSGVHSDIQ MTQSPSSLSA SVGDRVTITC STSSSVSYMH WYQQKPGKAP _CD28_KLLIYSTSNL ASGVPSRFSG SGSGTDFTFT ISSLQPEDIA TYYCHQWSSY CD40PTFGQGTKVE IKRAAAGSGG SGILVKQSPM LVAYDNAVNL SCKYSYNLFSREFRASLHKG LDSAVEVCVV YGNYSQQLQV YSKTGFNCDG KLGNESVTFYLQNLYVNQTD IYFCKIEVMY PPPYLDNEKS NGTIIHVKGK HLCPSPLFPGPSKPFWVLVV VGGVLACYSL LVTVAFIIFW VRSKRSRLLH SDYMNMTPRRPGPTRKHYQP YAPPRDFAAY RSKKVAKKPT NKAPHPKQEP QEINFPDDLPGSNTAAPVQE TLHGCQPVTQ EDGKESRISV QERQ 120 OSM_BW431MGVLLTQRTL LSLVLALLFP SMASMQLQES GPGLVRPSQT LSLTCTVSGF /26TISSGYSWHW VRQPPGRGLE WIGYIQYSGI TNYNPSLKSR VTMLVDTSKN _spCD8QFSLRLSSVT AADTAVYYCA REDYDYHWYF DVWGQGSLVT VSSGGGGSGG _CD28_GGSGGGGSGV HSDIQMTQSP SSLSASVGDR VTITCSTSSS VSYMHWYQQK CD40PGKAPKLLIY STSNLASGVP SRFSGSGSGT DFTFTISSLQ PEDIATYYCHQWSSYPTFGQ GTKVEIKRAA AGSGGSGFVP VFLPAKPTTT PAPRPPTPAPTIASQPLSLR PEACRPAAGG AVHTRGLDFA CDIYIWAPLA GTCGVLLLSLVITLYCNHRN RSKRSRLLHS DYMNMTPRRP GPTRKHYQPY APPRDFAAYRSKKVAKKPTN KAPHPKQEPQ EINFPDDLPG SNTAAPVQET LHGCQPVTQE DGKESRISVQ ERQ121 CD8a_BW43 MALPVTALLL PLALLLHAAR PQLQESGPGL VRPSQTLSLT CTVSGFTISS1/26 GYSWHWVRQP PGRGLEWIGY IQYSGITNYN PSLKSRVTML VDTSKNQFSL _spCD8RLSSVTAADT AVYYCAREDY DYHWYFDVWG QGSLVTVSSG GGGSGGGGSG _CD28_GGGSGVHSDI QMTQSPSSLS ASVGDRVTIT CSTSSSVSYM HWYQQKPGKA CD40PKLLIYSTSN LASGVPSRFS GSGSGTDFTF TISSLQPEDI ATYYCHQWSSYPTFGQGTKV EIKRAAAGSG GSGFVPVFLP AKPTTTPAPR PPTPAPTIASQPLSLRPEAC RPAAGGAVHT RGLDFACDIY IWAPLAGTCG VLLLSLVITLYCNHRNRSKR SRLLHSDYMN MTPRRPGPTR KHYQPYAPPR DFAAYRSKKVAKKPTNKAPH PKQEPQEINF PDDLPGSNTA APVQETLHGC QPVTQEDGKE SRISVQERQ 122CD2_BW431 MSFPCKFVAS FLLIFNVSSK GAVSQLQESG PGLVRPSQTL SLTCTVSGFT /26ISSGYSWHWV RQPPGRGLEW IGYIQYSGIT NYNPSLKSRV TMLVDTSKNQ _spCD8FSLRLSSVTA ADTAVYYCAR EDYDYHWYFD VWGQGSLVTV SSGGGGSGGG _CD28_GSGGGGSGVH SDIQMTQSPS SLSASVGDRV TITCSTSSSV SYMHWYQQKP CD40GKAPKLLIYS TSNLASGVPS RFSGSGSGTD FTFTISSLQP EDIATYYCHQWSSYPTFGQG TKVEIKRAAA GSGGSGFVPV FLPAKPTTTP APRPPTPAPTIASQPLSLRP EACRPAAGGA VHTRGLDFAC DIYIWAPLAG TCGVLLLSLVITLYCNHRNR SKRSRLLHSD YMNMTPRRPG PTRKHYQPYA PPRDFAAYRSKKVAKKPTNK APHPKQEPQE INFPDDLPGS NTAAPVQETL HGCQPVTQED GKESRISVQE RQ 123IL2_BW431 MYRMQLLSCI ALSLALVTNS QLQESGPGLV RPSQTLSLTC TVSGFTISSG /26YSWHWVRQPP GRGLEWIGYI QYSGITNYNP SLKSRVTMLV DTSKNQFSLR _spCD8LSSVTAADTA VYYCAREDYD YHWYFDVWGQ GSLVTVSSGG GGSGGGGSGG _CD28_CD4GGSGVHSDIQ MTQSPSSLSA SVGDRVTITC STSSSVSYMH WYQQKPGKAP 0KLLIYSTSNL ASGVPSRFSG SGSGTDFTFT ISSLQPEDIA TYYCHQWSSYPTFGQGTKVE IKRAAAGSGG SGFVPVFLPA KPTTTPAPRP PTPAPTIASQPLSLRPEACR PAAGGAVHTR GLDFACDIYI WAPLAGTCGV LLLSLVITLYCNHRNRSKRS RLLHSDYMNM TPRRPGPTRK HYQPYAPPRD FAAYRSKKVAKKPTNKAPHP KQEPQEINFP DDLPGSNTAA PVQETLHGCQ PVTQEDGKES RISVQERQ 124 GM-MWLQSLLLLG TVACSISQLQ ESGPGLVRPS QTLSLTCTVS GFTISSGYSW CSF_BW431HWVRQPPGRG LEWIGYIQYS GITNYNPSLK SRVTMLVDTS KNQFSLRLSS /26VTAADTAVYY CAREDYDYHW YFDVWGQGSL VTVSSGGGGS GGGGSGGGGS _spCD8GVHSDIQMTQ SPSSLSASVG DRVTITCSTS SSVSYMHWYQ QKPGKAPKLL _CD28_CD4IYSTSNLASG VPSRFSGSGS GTDFTFTISS LQPEDIATYY CHQWSSYPTF 0GQGTKVEIKR AAAGSGGSGF VPVFLPAKPT TTPAPRPPTP APTIASQPLSLRPEACRPAA GGAVHTRGLD FACDIYIWAP LAGTCGVLLL SLVITLYCNHRNRSKRSRLL HSDYMNMTPR RPGPTRKHYQ PYAPPRDFAA YRSKKVAKKPTNKAPHPKQE PQEINFPDDL PGSNTAAPVQ ETLHGCQPVT QEDGKESRIS VQERQ 125 hIgGk-MEAPAQLLFL LLLWLPDTTR QLQESGPGLV RPSQTLSLTC TVSGFTISSG VIII_BW43YSWHWVRQPP GRGLEWIGYI QYSGITNYNP SLKSRVTMLV DTSKNQFSLR 1/26LSSVTAADTA VYYCAREDYD YHWYFDVWGQ GSLVTVSSGG GGSGGGGSGG _spCD8GGSGVHSDIQ MTQSPSSLSA SVGDRVTITC STSSSVSYMH WYQQKPGKAP _CD28_KLLIYSTSNL ASGVPSRFSG SGSGTDFTFT ISSLQPEDIA TYYCHQWSSY CD40PTFGQGTKVE IKRAAAGSGG SGFVPVFLPA KPTTTPAPRP PTPAPTIASQPLSLRPEACR PAAGGAVHTR GLDFACDIYI WAPLAGTCGV LLLSLVITLYCNHRNRSKRS RLLHSDYMNM TPRRPGPTRK HYQPYAPPRD FAAYRSKKVAKKPTNKAPHP KQEPQEINFP DDLPGSNTAA PVQETLHGCQ PVTQEDGKES RISVQERQ 126OSM_BW431 MGVLLTQRTL LSLVLALLFP SMASMQLQES GPGLVRPSQT LSLTCTVSGF /26TISSGYSWHW VRQPPGRGLE WIGYIQYSGI TNYNPSLKSR VTMLVDTSKN _CD28TMQFSLRLSSVT AADTAVYYCA REDYDYHWYF DVWGQGSLVT VSSGGGGSGG _CD28_GGSGGGGSGV HSDIQMTQSP SSLSASVGDR VTITCSTSSS VSYMHWYQQK CD40PGKAPKLLIY STSNLASGVP SRFSGSGSGT DFTFTISSLQ PEDIATYYCHQWSSYPTFGQ GTKVEIKRAA AGSGGSGFWV LVVVGGVLAC YSLLVTVAFIIFWVRSKRSR LLHSDYMNMT PRRPGPTRKH YQPYAPPRDF AAYRSKKVAKKPTNKAPHPK QEPQEINFPD DLPGSNTAAP VQETLHGCQP VTQEDGKESR ISVQERQ 127CD8a_BW43 MALPVTALLL PLALLLHAAR PQLQESGPGL VRPSQTLSLT CTVSGFTISS 1/26GYSWHWVRQP PGRGLEWIGY IQYSGITNYN PSLKSRVTML VDTSKNQFSL _CD28TMRLSSVTAADT AVYYCAREDY DYHWYFDVWG QGSLVTVSSG GGGSGGGGSG _CD28_GGGSGVHSDI QMTQSPSSLS ASVGDRVTIT CSTSSSVSYM HWYQQKPGKA CD40PKLLIYSTSN LASGVPSRFS GSGSGTDFTF TISSLQPEDI ATYYCHQWSSYPTFGQGTKV EIKRAAAGSG GSGFWVLVVV GGVLACYSLL VTVAFIIFWVRSKRSRLLHS DYMNMTPRRP GPTRKHYQPY APPRDFAAYR SKKVAKKPTNKAPHPKQEPQ EINFPDDLPG SNTAAPVQET LHGCQPVTQE DGKESRISVQ ERQ 128 CD2_BW431MSFPCKFVAS FLLIFNVSSK GAVSQLQESG PGLVRPSQTL SLTCTVSGFT /26ISSGYSWHWV RQPPGRGLEW IGYIQYSGIT NYNPSLKSRV TMLVDTSKNQ _CD28TMFSLRLSSVTA ADTAVYYCAR EDYDYHWYFD VWGQGSLVTV SSGGGGSGGG _CD28_GSGGGGSGVH SDIQMTQSPS SLSASVGDRV TITCSTSSSV SYMHWYQQKP CD40GKAPKLLIYS TSNLASGVPS RFSGSGSGTD FTFTISSLQP EDIATYYCHQWSSYPTFGQG TKVEIKRAAA GSGGSGFWVL VVVGGVLACY SLLVTVAFIIFWVRSKRSRL LHSDYMNMTP RRPGPTRKHY QPYAPPRDFA AYRSKKVAKKPTNKAPHPKQ EPQEINFPDD LPGSNTAAPV QETLHGCQPV TQEDGKESRI SVQERQ 129IL2_BW431 MYRMQLLSCI ALSLALVTNS QLQESGPGLV RPSQTLSLTC TVSGFTISSG /26YSWHWVRQPP GRGLEWIGYI QYSGITNYNP SLKSRVTMLV DTSKNQFSLR _CD28TMLSSVTAADTA VYYCAREDYD YHWYFDVWGQ GSLVTVSSGG GGSGGGGSGG _CD28_GGSGVHSDIQ MTQSPSSLSA SVGDRVTITC STSSSVSYMH WYQQKPGKAP CD40KLLIYSTSNL ASGVPSRFSG SGSGTDFTFT ISSLQPEDIA TYYCHQWSSYPTFGQGTKVE IKRAAAGSGG SGFWVLVVVG GVLACYSLLV TVAFIIFWVRSKRSRLLHSD YMNMTPRRPG PTRKHYQPYA PPRDFAAYRS KKVAKKPTNKAPHPKQEPQE INFPDDLPGS NTAAPVQETL HGCQPVTQED GKESRISVQE RQ 130 GM-CSFMWLQSLLLLG TVACSISQLQ ESGPGLVRPS QTLSLTCTVS GFTISSGYSW _BW431/26HWVRQPPGRG LEWIGYIQYS GITNYNPSLK SRVTMLVDTS KNQFSLRLSS _CD28TMVTAADTAVYY CAREDYDYHW YFDVWGQGSL VTVSSGGGGS GGGGSGGGGS _CD28_GVHSDIQMTQ SPSSLSASVG DRVTITCSTS SSVSYMHWYQ QKPGKAPKLL CD40IYSTSNLASG VPSRFSGSGS GTDFTFTISS LQPEDIATYY CHQWSSYPTFGQGTKVEIKR AAAGSGGSGF WVLVVVGGVL ACYSLLVTVA FIIFWVRSKRSRLLHSDYMN MTPRRPGPTR KHYQPYAPPR DFAAYRSKKV AKKPTNKAPHPKQEPQEINF PDDLPGSNTA APVQETLHGC QPVTQEDGKE SRISVQERQ 131 hIgGk-MEAPAQLLFL LLLWLPDTTR QLQESGPGLV RPSQTLSLTC TVSGFTISSG VIIIYSWHWVRQPP GRGLEWIGYI QYSGITNYNP SLKSRVTMLV DTSKNQFSLR _BW431/26LSSVTAADTA VYYCAREDYD YHWYFDVWGQ GSLVTVSSGG GGSGGGGSGG _CD28TMGGSGVHSDIQ MTQSPSSLSA SVGDRVTITC STSSSVSYMH WYQQKPGKAP _CD28_KLLIYSTSNL ASGVPSRFSG SGSGTDFTFT ISSLQPEDIA TYYCHQWSSY CD40PTFGQGTKVE IKRAAAGSGG SGFWVLVVVG GVLACYSLLV TVAFIIFWVRSKRSRLLHSD YMNMTPRRPG PTRKHYQPYA PPRDFAAYRS KKVAKKPTNKAPHPKQEPQE INFPDDLPGS NTAAPVQETL HGCQPVTQED GKESRISVQE RQ 132 OSM_HuT84.MGVLLTQRTL LSLVLALLFP SMASMEVQLV ESGGGLVQPG GSLRLSCAAS 66GFNIKDTYMH WVRQAPGKGL EWVARIDPAN GNSKYADSVK GRFTISADTS _spCD28KNTAYLQMNS LRAEDTAVYY CAPFGYYVSD YAMAYWGQGT LVTVSSGGGG _CD28_SGGGGSGGGG SDIQLTQSPS SLSASVGDRV TITCRAGESV DIFGVGFLHW CD40YQQKPGKAPK LLIYRASNLE SGVPSRFSGS GSRTDFTLTI SSLQPEDFAT CTP223YYCQQTNEDP YTFGQGTKVE IKAAAGSGGS GILVKQSPML VAYDNAVNLSCKYSYNLFSR EFRASLHKGL DSAVEVCVVY GNYSQQLQVY SKTGFNCDGKLGNESVTFYL QNLYVNQTDI YFCKIEVMYP PPYLDNEKSN GTIIHVKGKHLCPSPLFPGP SKPFWVLVVV GGVLACYSLL VTVAFIIFWV RSKRSRLLHSDYMNMTPRRP GPTRKHYQPY APPRDFAAYR SKKVAKKPTN KAPHPKQEPQEINFPDDLPG SNTAAPVQET LHGCQPVTQE DGKESRISVQ ERQ 133 CD8a_HuT8MALPVTALLL PLALLLHAAR PEVQLVESGG GLVQPGGSLR LSCAASGFNI 4.66KDTYMHWVRQ APGKGLEWVA RIDPANGNSK YADSVKGRFT ISADTSKNTA _spCD28YLQMNSLRAE DTAVYYCAPF GYYVSDYAMA YWGQGTLVTV SSGGGGSGGG _CD28_GSGGGGSDIQ LTQSPSSLSA SVGDRVTITC RAGESVDIFG VGFLHWYQQK CD40PGKAPKLLIY RASNLESGVP SRFSGSGSRT DFTLTISSLQ PEDFATYYCQQTNEDPYTFG QGTKVEIKAA AGSGGSGILV KQSPMLVAYD NAVNLSCKYSYNLFSREFRA SLHKGLDSAV EVCVVYGNYS QQLQVYSKTG FNCDGKLGNESVTFYLQNLY VNQTDIYFCK IEVMYPPPYL DNEKSNGTII HVKGKHLCPSPLFPGPSKPF WVLVVVGGVL ACYSLLVTVA FIIFWVRSKR SRLLHSDYMNMTPRRPGPTR KHYQPYAPPR DFAAYRSKKV AKKPTNKAPH PKQEPQEINFPDDLPGSNTA APVQETLHGC QPVTQEDGKE SRISVQERQ 134 CD2_HuT84.MSFPCKFVAS FLLIFNVSSK GAVSEVQLVE SGGGLVQPGG SLRLSCAASG 66FNIKDTYMHW VRQAPGKGLE WVARIDPANG NSKYADSVKG RFTISADTSK _spCD28NTAYLQMNSL RAEDTAVYYC APFGYYVSDY AMAYWGQGTL VTVSSGGGGS _CD28_GGGGSGGGGS DIQLTQSPSS LSASVGDRVT ITCRAGESVD IFGVGFLHWY CD40QQKPGKAPKL LIYRASNLES GVPSRFSGSG SRTDFTLTIS SLQPEDFATYYCQQTNEDPY TFGQGTKVEI KAAAGSGGSG ILVKQSPMLV AYDNAVNLSCKYSYNLFSRE FRASLHKGLD SAVEVCVVYG NYSQQLQVYS KTGFNCDGKLGNESVTFYLQ NLYVNQTDIY FCKIEVMYPP PYLDNEKSNG TIIHVKGKHLCPSPLFPGPS KPFWVLVVVG GVLACYSLLV TVAFIIFWVR SKRSRLLHSDYMNMTPRRPG PTRKHYQPYA PPRDFAAYRS KKVAKKPTNK APHPKQEPQEINFPDDLPGS NTAAPVQETL HGCQPVTQED GKESRISVQE RQ 135 IL2_HuT84.MYRMQLLSCI ALSLALVTNS EVQLVESGGG LVQPGGSLRL SCAASGFNIK 66DTYMHWVRQA PGKGLEWVAR IDPANGNSKY ADSVKGRFTI SADTSKNTAY _spCD28LQMNSLRAED TAVYYCAPFG YYVSDYAMAY WGQGTLVTVS SGGGGSGGGG _CD28_SGGGGSDIQL TQSPSSLSAS VGDRVTITCR AGESVDIFGV GFLHWYQQKP CD40GKAPKLLIYR ASNLESGVPS RFSGSGSRTD FTLTISSLQP EDFATYYCQQTNEDPYTFGQ GTKVEIKAAA GSGGSGILVK QSPMLVAYDN AVNLSCKYSYNLFSREFRAS LHKGLDSAVE VCVVYGNYSQ QLQVYSKTGF NCDGKLGNESVTFYLQNLYV NQTDIYFCKI EVMYPPPYLD NEKSNGTIIH VKGKHLCPSPLFPGPSKPFW VLVVVGGVLA CYSLLVTVAF IIFWVRSKRS RLLHSDYMNMTPRRPGPTRK HYQPYAPPRD FAAYRSKKVA KKPTNKAPHP KQEPQEINFPDDLPGSNTAA PVQETLHGCQ PVTQEDGKES RISVQERQ 136 GM-MWLQSLLLLG TVACSISEVQ LVESGGGLVQ PGGSLRLSCA ASGFNIKDTY CSF_HuT84.MHWVRQAPGK GLEWVARIDP ANGNSKYADS VKGRFTISAD TSKNTAYLQM 66NSLRAEDTAV YYCAPFGYYV SDYAMAYWGQ GTLVTVSSGG GGSGGGGSGG _spCD28GGSDIQLTQS PSSLSASVGD RVTITCRAGE SVDIFGVGFL HWYQQKPGKA _CD28_PKLLIYRASN LESGVPSRFS GSGSRTDFTL TISSLQPEDF ATYYCQQTNE CD40DPYTFGQGTK VEIKAAAGSG GSGILVKQSP MLVAYDNAVN LSCKYSYNLFSREFRASLHK GLDSAVEVCV VYGNYSQQLQ VYSKTGFNCD GKLGNESVTFYLQNLYVNQT DIYFCKIEVM YPPPYLDNEK SNGTIIHVKG KHLCPSPLFPGPSKPFWVLV VVGGVLACYS LLVTVAFIIF WVRSKRSRLL HSDYMNMTPRRPGPTRKHYQ PYAPPRDFAA YRSKKVAKKP TNKAPHPKQE PQEINFPDDLPGSNTAAPVQ ETLHGCQPVT QEDGKESRIS VQERQ 137 hIgGk-MEAPAQLLFL LLLWLPDTTR EVQLVESGGG LVQPGGSLRL SCAASGFNIK VIII_HuT8DTYMHWVRQA PGKGLEWVAR IDPANGNSKY ADSVKGRFTI SADTSKNTAY 4.66LQMNSLRAED TAVYYCAPFG YYVSDYAMAY WGQGTLVTVS SGGGGSGGGG _spCD28SGGGGSDIQL TQSPSSLSAS VGDRVTITCR AGESVDIFGV GFLHWYQQKP _CD28_GKAPKLLIYR ASNLESGVPS RFSGSGSRTD FTLTISSLQP EDFATYYCQQ CD40TNEDPYTFGQ GTKVEIKAAA GSGGSGILVK QSPMLVAYDN AVNLSCKYSYNLFSREFRAS LHKGLDSAVE VCVVYGNYSQ QLQVYSKTGF NCDGKLGNESVTFYLQNLYV NQTDIYFCKI EVMYPPPYLD NEKSNGTIIH VKGKHLCPSPLFPGPSKPFW VLVVVGGVLA CYSLLVTVAF IIFWVRSKRS RLLHSDYMNMTPRRPGPTRK HYQPYAPPRD FAAYRSKKVA KKPTNKAPHP KQEPQEINFPDDLPGSNTAA PVQETLHGCQ PVTQEDGKES RISVQERQ 138 OSM_HuT84MGVLLTQRTL LSLVLALLFP SMASMEVQLV ESGGGLVQPG GSLRLSCAAS .6 6_spCD8GFNIKDTYMH WVRQAPGKGL EWVARIDPAN GNSKYADSVK GRFTISADTS _CD28_CD4KNTAYLQMNS LRAEDTAVYY CAPFGYYVSD YAMAYWGQGT LVTVSSGGGG 0SGGGGSGGGG SDIQLTQSPS SLSASVGDRV TITCRAGESV DIFGVGFLHWYQQKPGKAPK LLIYRASNLE SGVPSRFSGS GSRTDFTLTI SSLQPEDFATYYCQQTNEDP YTFGQGTKVE IKAAAGSGGS GFVPVFLPAK PTTTPAPRPPTPAPTIASQP LSLRPEACRP AAGGAVHTRG LDFACDIYIW APLAGTCGVLLLSLVITLYC NHRNRSKRSR LLHSDYMNMT PRRPGPTRKH YQPYAPPRDFAAYRSKKVAK KPTNKAPHPK QEPQEINFPD DLPGSNTAAP VQETLHGCQPVTQEDGKESR ISVQERQ 139 CD8a_HuT8MALPVTALLL PLALLLHAAR PEVQLVESGG GLVQPGGSLR LSCAASGFNI 4.66KDTYMHWVRQ APGKGLEWVA RIDPANGNSK YADSVKGRFT ISADTSKNTA _spCD8YLQMNSLRAE DTAVYYCAPF GYYVSDYAMA YWGQGTLVTV SSGGGGSGGG _CD28_GSGGGGSDIQ LTQSPSSLSA SVGDRVTITC RAGESVDIFG VGFLHWYQQK CD40PGKAPKLLIY RASNLESGVP SRFSGSGSRT DFTLTISSLQ PEDFATYYCQQTNEDPYTFG QGTKVEIKAA AGSGGSGFVP VFLPAKPTTT PAPRPPTPAPTIASQPLSLR PEACRPAAGG AVHTRGLDFA CDIYIWAPLA GTCGVLLLSLVITLYCNHRN RSKRSRLLHS DYMNMTPRRP GPTRKHYQPY APPRDFAAYRSKKVAKKPTN KAPHPKQEPQ EINFPDDLPG SNTAAPVQET LHGCQPVTQE DGKESRISVQ ERQ140 CD2_HuT84 MSFPCKFVAS FLLIFNVSSK GAVSEVQLVE SGGGLVQPGG SLRLSCAASG.66_spCD8 FNIKDTYMHW VRQAPGKGLE WVARIDPANG NSKYADSVKG RFTISADTSK _CD28_NTAYLQMNSL RAEDTAVYYC APFGYYVSDY AMAYWGQGTL VTVSSGGGGS CD40GGGGSGGGGS DIQLTQSPSS LSASVGDRVT ITCRAGESVD IFGVGFLHWYQQKPGKAPKL LIYRASNLES GVPSRFSGSG SRTDFTLTIS SLQPEDFATYYCQQTNEDPY TFGQGTKVEI KAAAGSGGSG FVPVFLPAKP TTTPAPRPPTPAPTIASQPL SLRPEACRPA AGGAVHTRGL DFACDIYIWA PLAGTCGVLLLSLVITLYCN HRNRSKRSRL LHSDYMNMTP RRPGPTRKHY QPYAPPRDFAAYRSKKVAKK PTNKAPHPKQ EPQEINFPDD LPGSNTAAPV QETLHGCQPV TQEDGKESRI SVQERQ141 IL2_HuT84 MYRMQLLSCI ALSLALVTNS EVQLVESGGG LVQPGGSLRL SCAASGFNIK. 66 DTYMHWVRQA PGKGLEWVAR IDPANGNSKY ADSVKGRFTI SADTSKNTAY _spCD8LQMNSLRAED TAVYYCAPFG YYVSDYAMAY WGQGTLVTVS SGGGGSGGGG _CD28_CD4SGGGGSDIQL TQSPSSLSAS VGDRVTITCR AGESVDIFGV GFLHWYQQKP 0GKAPKLLIYR ASNLESGVPS RFSGSGSRTD FTLTISSLQP EDFATYYCQQTNEDPYTFGQ GTKVEIKAAA GSGGSGFVPV FLPAKPTTTP APRPPTPAPTIASQPLSLRP EACRPAAGGA VHTRGLDFAC DIYIWAPLAG TCGVLLLSLVITLYCNHRNR SKRSRLLHSD YMNMTPRRPG PTRKHYQPYA PPRDFAAYRSKKVAKKPTNK APHPKQEPQE INFPDDLPGS NTAAPVQETL HGCQPVTQED GKESRISVQE RQ 142GM- MWLQSLLLLG TVACSISEVQ LVESGGGLVQ PGGSLRLSCA ASGFNIKDTY CSF_HuT84.MHWVRQAPGK GLEWVARIDP ANGNSKYADS VKGRFTISAD TSKNTAYLQM 66NSLRAEDTAV YYCAPFGYYV SDYAMAYWGQ GTLVTVSSGG GGSGGGGSGG _spCD8GGSDIQLTQS PSSLSASVGD RVTITCRAGE SVDIFGVGFL HWYQQKPGKA _CD28_PKLLIYRASN LESGVPSRFS GSGSRTDFTL TISSLQPEDF ATYYCQQTNE CD40DPYTFGQGTK VEIKAAAGSG GSGFVPVFLP AKPTTTPAPR PPTPAPTIASQPLSLRPEAC RPAAGGAVHT RGLDFACDIY IWAPLAGTCG VLLLSLVITLYCNHRNRSKR SRLLHSDYMN MTPRRPGPTR KHYQPYAPPR DFAAYRSKKVAKKPTNKAPH PKQEPQEINF PDDLPGSNTA APVQETLHGC QPVTQEDGKE SRISVQERQ 143hIgGk- MEAPAQLLFL LLLWLPDTTR EVQLVESGGG LVQPGGSLRL SCAASGFNIK VIII_HuT8DTYMHWVRQA PGKGLEWVAR IDPANGNSKY ADSVKGRFTI SADTSKNTAY 4.66LQMNSLRAED TAVYYCAPFG YYVSDYAMAY WGQGTLVTVS SGGGGSGGGG _spCD8SGGGGSDIQL TQSPSSLSAS VGDRVTITCR AGESVDIFGV GFLHWYQQKP _CD28_GKAPKLLIYR ASNLESGVPS RFSGSGSRTD FTLTISSLQP EDFATYYCQQ CD40TNEDPYTFGQ GTKVEIKAAA GSGGSGFVPV FLPAKPTTTP APRPPTPAPTIASQPLSLRP EACRPAAGGA VHTRGLDFAC DIYIWAPLAG TCGVLLLSLVITLYCNHRNR SKRSRLLHSD YMNMTPRRPG PTRKHYQPYA PPRDFAAYRSKKVAKKPTNK APHPKQEPQE INFPDDLPGS NTAAPVQETL HGCQPVTQED GKESRISVQE RQ 144OSM_HuT84. MGVLLTQRTL LSLVLALLFP SMASMEVQLV ESGGGLVQPG GSLRLSCAAS 66GFNIKDTYMH WVRQAPGKGL EWVARIDPAN GNSKYADSVK GRFTISADTS _CD28TMKNTAYLQMNS LRAEDTAVYY CAPFGYYVSD YAMAYWGQGT LVTVSSGGGG _CD28_SGGGGSGGGG SDIQLTQSPS SLSASVGDRV TITCRAGESV DIFGVGFLHW CD40YQQKPGKAPK LLIYRASNLE SGVPSRFSGS GSRTDFTLTI SSLQPEDFATYYCQQTNEDP YTFGQGTKVE IKAAAGSGGS GFWVLVVVGG VLACYSLLVTVAFIIFWVRS KRSRLLHSDY MNMTPRRPGP TRKHYQPYAP PRDFAAYRSKKVAKKPTNKA PHPKQEPQEI NFPDDLPGSN TAAPVQETLH GCQPVTQEDG KESRISVQER Q 145CD8a_HuT8 MALPVTALLL PLALLLHAAR PEVQLVESGG GLVQPGGSLR LSCAASGFNI 4.66KDTYMHWVRQ APGKGLEWVA RIDPANGNSK YADSVKGRFT ISADTSKNTA _CD28TMYLQMNSLRAE DTAVYYCAPF GYYVSDYAMA YWGQGTLVTV SSGGGGSGGG _CD28_GSGGGGSDIQ LTQSPSSLSA SVGDRVTITC RAGESVDIFG VGFLHWYQQK CD40PGKAPKLLIY RASNLESGVP SRFSGSGSRT DFTLTISSLQ PEDFATYYCQQTNEDPYTFG QGTKVEIKAA AGSGGSGFWV LVVVGGVLAC YSLLVTVAFIIFWVRSKRSR LLHSDYMNMT PRRPGPTRKH YQPYAPPRDF AAYRSKKVAKKPTNKAPHPK QEPQEINFPD DLPGSNTAAP VQETLHGCQP VTQEDGKESR ISVQERQ 146CD2_HuT84. MSFPCKFVAS FLLIFNVSSK GAVSEVQLVE SGGGLVQPGG SLRLSCAASG 66FNIKDTYMHW VRQAPGKGLE WVARIDPANG NSKYADSVKG RFTISADTSK _CD28TMNTAYLQMNSL RAEDTAVYYC APFGYYVSDY AMAYWGQGTL VTVSSGGGGS _CD28_GGGGSGGGGS DIQLTQSPSS LSASVGDRVT ITCRAGESVD IFGVGFLHWY CD40QQKPGKAPKL LIYRASNLES GVPSRFSGSG SRTDFTLTIS SLQPEDFATYYCQQTNEDPY TFGQGTKVEI KAAAGSGGSG FWVLVVVGGV LACYSLLVTVAFIIFWVRSK RSRLLHSDYM NMTPRRPGPT RKHYQPYAPP RDFAAYRSKKVAKKPTNKAP HPKQEPQEIN FPDDLPGSNT AAPVQETLHG CQPVTQEDGK ESRISVQERQ 147IL2_HuT84. MYRMQLLSCI ALSLALVTNS EVQLVESGGG LVQPGGSLRL SCAASGFNIK 66DTYMHWVRQA PGKGLEWVAR IDPANGNSKY ADSVKGRFTI SADTSKNTAY _CD28TMLQMNSLRAED TAVYYCAPFG YYVSDYAMAY WGQGTLVTVS SGGGGSGGGG _CD28_SGGGGSDIQL TQSPSSLSAS VGDRVTITCR AGESVDIFGV GFLHWYQQKP CD40GKAPKLLIYR ASNLESGVPS RFSGSGSRTD FTLTISSLQP EDFATYYCQQTNEDPYTFGQ GTKVEIKAAA GSGGSGFWVL VVVGGVLACY SLLVTVAFIIFWVRSKRSRL LHSDYMNMTP RRPGPTRKHY QPYAPPRDFA AYRSKKVAKKPTNKAPHPKQ EPQEINFPDD LPGSNTAAPV QETLHGCQPV TQEDGKESRI SVQERQ 148 GM-MWLQSLLLLG TVACSISEVQ LVESGGGLVQ PGGSLRLSCA ASGFNIKDTY CSF_HuT84.MHWVRQAPGK GLEWVARIDP ANGNSKYADS VKGRFTISAD TSKNTAYLQM 66NSLRAEDTAV YYCAPFGYYV SDYAMAYWGQ GTLVTVSSGG GGSGGGGSGG _CD28TMGGSDIQLTQS PSSLSASVGD RVTITCRAGE SVDIFGVGFL HWYQQKPGKA _CD28_PKLLIYRASN LESGVPSRFS GSGSRTDFTL TISSLQPEDF ATYYCQQTNE CD40DPYTFGQGTK VEIKAAAGSG GSGFWVLVVV GGVLACYSLL VTVAFIIFWVRSKRSRLLHS DYMNMTPRRP GPTRKHYQPY APPRDFAAYR SKKVAKKPTNKAPHPKQEPQ EINFPDDLPG SNTAAPVQET LHGCQPVTQE DGKESRISVQ ERQ 149 hIgGk-MEAPAQLLFL LLLWLPDTTR EVQLVESGGG LVQPGGSLRL SCAASGFNIK VIII_HuT8DTYMHWVRQA PGKGLEWVAR IDPANGNSKY ADSVKGRFTI SADTSKNTAY 4.66LQMNSLRAED TAVYYCAPFG YYVSDYAMAY WGQGTLVTVS SGGGGSGGGG _CD28TMSGGGGSDIQL TQSPSSLSAS VGDRVTITCR AGESVDIFGV GFLHWYQQKP _CD28_GKAPKLLIYR ASNLESGVPS RFSGSGSRTD FTLTISSLQP EDFATYYCQQ CD40TNEDPYTFGQ GTKVEIKAAA GSGGSGFWVL VVVGGVLACY SLLVTVAFIIFWVRSKRSRL LHSDYMNMTP RRPGPTRKHY QPYAPPRDFA AYRSKKVAKKPTNKAPHPKQ EPQEINFPDD LPGSNTAAPV QETLHGCQPV TQEDGKESRI SVQERQ 150OSM_MFE23 MGVLLTQRTL LSLVLALLFP SMASMQVKLQ QSGAELVRSG TSVKLSCTAS_spCD28(t GFNIKDSYMH WLRQGPEQGL EWIGWIDPEN GDTEYAPKFQ GKATFTTDTS run)SNTAYLQLSS LTSEDTAVYY CNEGTPTGPY YFDYWGQGTT VTVSSGGGGS _CD28_GGGGSGGGGS ENVLTQSPAI MSASPGEKVT ITCSASSSVS YMHWFQQKPG CD40TSPKLWIYST SNLASGVPAR FSGSGSGTSY SLTISRMEAE DAATYYCQQRSSYPLTFGAG TKLELKRAAA GSGGSGIIHV KGKHLCPSPL FPGPSKPFWVLVVVGGVLAC YSLLVTVAFI IFWVRSKRSR LLHSDYMNMT PRRPGPTRKHYQPYAPPRDF AAYRSKKVAK KPTNKAPHPK QEPQEINFPD DLPGSNTAAPVQETLHGCQP VTQEDGKESR ISVQERQ 151 CD8a_MFE2MALPVTALLL PLALLLHAAR PQVKLQQSGA ELVRSGTSVK LSCTASGFNI 3KDSYMHWLRQ GPEQGLEWIG WIDPENGDTE YAPKFQGKAT FTTDTSSNTA _spCD28(tYLQLSSLTSE DTAVYYCNEG TPTGPYYFDY WGQGTTVTVS SGGGGSGGGG run)SGGGGSENVL TQSPAIMSAS PGEKVTITCS ASSSVSYMHW FQQKPGTSPK _CD28_LWIYSTSNLA SGVPARFSGS GSGTSYSLTI SRMEAEDAAT YYCQQRSSYP CD40LTFGAGTKLE LKRAAAGSGG SGIIHVKGKH LCPSPLFPGP SKPFWVLVVVGGVLACYSLL VTVAFIIFWV RSKRSRLLHS DYMNMTPRRP GPTRKHYQPYAPPRDFAAYR SKKVAKKPTN KAPHPKQEPQ EINFPDDLPG SNTAAPVQETLHGCQPVTQE DGKESRISVQ ERQ 152 CD2_MFE23MSFPCKFVAS FLLIFNVSSK GAVSQVKLQQ SGAELVRSGT SVKLSCTASG _spCD28(tFNIKDSYMHW LRQGPEQGLE WIGWIDPENG DTEYAPKFQG KATFTTDTSS run)NTAYLQLSSL TSEDTAVYYC NEGTPTGPYY FDYWGQGTTV TVSSGGGGSG _CD28_GGGSGGGGSE NVLTQSPAIM SASPGEKVTI TCSASSSVSY MHWFQQKPGT CD40SPKLWIYSTS NLASGVPARF SGSGSGTSYS LTISRMEAED AATYYCQQRSSYPLTFGAGT KLELKRAAAG SGGSGIIHVK GKHLCPSPLF PGPSKPFWVLVVVGGVLACY SLLVTVAFII FWVRSKRSRL LHSDYMNMTP RRPGPTRKHYQPYAPPRDFA AYRSKKVAKK PTNKAPHPKQ EPQEINFPDD LPGSNTAAPVQETLHGCQPV TQEDGKESRI SVQERQ 153 IL2_MFE23MYRMQLLSCI ALSLALVTNS QVKLQQSGAE LVRSGTSVKL SCTASGFNIK _spCD28(tDSYMHWLRQG PEQGLEWIGW IDPENGDTEY APKFQGKATF TTDTSSNTAY run)LQLSSLTSED TAVYYCNEGT PTGPYYFDYW GQGTTVTVSS GGGGSGGGGS _CD28_GGGGSENVLT QSPAIMSASP GEKVTITCSA SSSVSYMHWF QQKPGTSPKL CD40WIYSTSNLAS GVPARFSGSG SGTSYSLTIS RMEAEDAATY YCQQRSSYPLTFGAGTKLEL KRAAAGSGGS GIIHVKGKHL CPSPLFPGPS KPFWVLVVVGGVLACYSLLV TVAFIIFWVR SKRSRLLHSD YMNMTPRRPG PTRKHYQPYAPPRDFAAYRS KKVAKKPTNK APHPKQEPQE INFPDDLPGS NTAAPVQETLHGCQPVTQED GKESRISVQE RQ 154 GM-MWLQSLLLLG TVACSISQVK LQQSGAELVR SGTSVKLSCT ASGFNIKDSY CSF_MFE23MHWLRQGPEQ GLEWIGWIDP ENGDTEYAPK FQGKATFTTD TSSNTAYLQL _spCD28(tSSLTSEDTAV YYCNEGTPTG PYYFDYWGQG TTVTVSSGGG GSGGGGSGGG run)GSENVLTQSP AIMSASPGEK VTITCSASSS VSYMHWFQQK PGTSPKLWIY _CD28_STSNLASGVP ARFSGSGSGT SYSLTISRME AEDAATYYCQ QRSSYPLTFG CD40AGTKLELKRA AAGSGGSGII HVKGKHLCPS PLFPGPSKPF WVLVVVGGVLACYSLLVTVA FIIFWVRSKR SRLLHSDYMN MTPRRPGPTR KHYQPYAPPRDFAAYRSKKV AKKPTNKAPH PKQEPQEINF PDDLPGSNTA APVQETLHGCQPVTQEDGKE SRISVQERQ 155 hIgGk-MEAPAQLLFL LLLWLPDTTR QVKLQQSGAE LVRSGTSVKL SCTASGFNIK VIII_MFE2DSYMHWLRQG PEQGLEWIGW IDPENGDTEY APKFQGKATF TTDTSSNTAY 3LQLSSLTSED TAVYYCNEGT PTGPYYFDYW GQGTTVTVSS GGGGSGGGGS _spCD28(tGGGGSENVLT QSPAIMSASP GEKVTITCSA SSSVSYMHWF QQKPGTSPKL run)WIYSTSNLAS GVPARFSGSG SGTSYSLTIS RMEAEDAATY YCQQRSSYPL _CD28_TFGAGTKLEL KRAAAGSGGS GIIHVKGKHL CPSPLFPGPS KPFWVLVVVG CD40GVLACYSLLV TVAFIIFWVR SKRSRLLHSD YMNMTPRRPG PTRKHYQPYAPPRDFAAYRS KKVAKKPTNK APHPKQEPQE INFPDDLPGS NTAAPVQETLHGCQPVTQED GKESRISVQE RQ 156 OSM_MFE23MGVLLTQRTL LSLVLALLFP SMASMQVQLQ QSGAELVRSG TSVKLSCTAS (K > Q)GFNIKDSYMH WLRQGPEQGL EWIGWIDPEN GDTEYAPKFQ GKATFTTDTS _spCD28(tSNTAYLQLSS LTSEDTAVYY CNEGTPTGPY YFDYWGQGTT VTVSSGGGGS run)GGGGSGGGGS ENVLTQSPAI MSASPGEKVT ITCSASSSVS YMHWFQQKPG _CD28_TSPKLWIYST SNLASGVPAR FSGSGSGTSY SLTISRMEAE DAATYYCQQR CD40SSYPLTFGAG TKLELKRAAA GSGGSGIIHV KGKHLCPSPL FPGPSKPFWVLVVVGGVLAC YSLLVTVAFI IFWVRSKRSR LLHSDYMNMT PRRPGPTRKHYQPYAPPRDF AAYRSKKVAK KPTNKAPHPK QEPQEINFPD DLPGSNTAAPVQETLHGCQP VTQEDGKESR ISVQERQ 157 CD8aMALPVTALLL PLALLLHAAR PQVQLQQSGA ELVRSGTSVK LSCTASGFNI _MFE2 3 (K >KDSYMHWLRQ GPEQGLEWIG WIDPENGDTE YAPKFQGKAT FTTDTSSNTA Q)YLQLSSLTSE DTAVYYCNEG TPTGPYYFDY WGQGTTVTVS SGGGGSGGGG _spCD28(tSGGGGSENVL TQSPAIMSAS PGEKVTITCS ASSSVSYMHW FQQKPGTSPK run)LWIYSTSNLA SGVPARFSGS GSGTSYSLTI SRMEAEDAAT YYCQQRSSYP _CD28_LTFGAGTKLE LKRAAAGSGG SGIIHVKGKH LCPSPLFPGP SKPFWVLVVV CD40GGVLACYSLL VTVAFIIFWV RSKRSRLLHS DYMNMTPRRP GPTRKHYQPYAPPRDFAAYR SKKVAKKPTN KAPHPKQEPQ EINFPDDLPG SNTAAPVQETLHGCQPVTQE DGKESRISVQ ERQ 158 CD2_MFE23MSFPCKFVAS FLLIFNVSSK GAVSQVQLQQ SGAELVRSGT SVKLSCTASG (K > Q)FNIKDSYMHW LRQGPEQGLE WIGWIDPENG DTEYAPKFQG KATFTTDTSS _spCD28(tNTAYLQLSSL TSEDTAVYYC NEGTPTGPYY FDYWGQGTTV TVSSGGGGSG run)GGGSGGGGSE NVLTQSPAIM SASPGEKVTI TCSASSSVSY MHWFQQKPGT _CD28_SPKLWIYSTS NLASGVPARF SGSGSGTSYS LTISRMEAED AATYYCQQRS CD40SYPLTFGAGT KLELKRAAAG SGGSGIIHVK GKHLCPSPLF PGPSKPFWVLVVVGGVLACY SLLVTVAFII FWVRSKRSRL LHSDYMNMTP RRPGPTRKHYQPYAPPRDFA AYRSKKVAKK PTNKAPHPKQ EPQEINFPDD LPGSNTAAPVQETLHGCQPV TQEDGKESRI SVQERQ 159 IL2_MFE23MYRMQLLSCI ALSLALVTNS QVQLQQSGAE LVRSGTSVKL SCTASGFNIK (K > Q)_spCDSYMHWLRQG PEQGLEWIGW IDPENGDTEY APKFQGKATF TTDTSSNTAY D28(trun)LQLSSLTSED TAVYYCNEGT PTGPYYFDYW GQGTTVTVSS GGGGSGGGGS _CD28_GGGGSENVLT QSPAIMSASP GEKVTITCSA SSSVSYMHWF QQKPGTSPKL CD40WIYSTSNLAS GVPARFSGSG SGTSYSLTIS RMEAEDAATY YCQQRSSYPLTFGAGTKLEL KRAAAGSGGS GIIHVKGKHL CPSPLFPGPS KPFWVLVVVGGVLACYSLLV TVAFIIFWVR SKRSRLLHSD YMNMTPRRPG PTRKHYQPYAPPRDFAAYRS KKVAKKPTNK APHPKQEPQE INFPDDLPGS NTAAPVQETLHGCQPVTQED GKESRISVQE RQ 160 GM-CSFMWLQSLLLLG TVACSISQVQ LQQSGAELVR SGTSVKLSCT ASGFNIKDSY _MFE23(K >MHWLRQGPEQ GLEWIGWIDP ENGDTEYAPK FQGKATFTTD TSSNTAYLQL Q)SSLTSEDTAV YYCNEGTPTG PYYFDYWGQG TTVTVSSGGG GSGGGGSGGG _spCD28(tGSENVLTQSP AIMSASPGEK VTITCSASSS VSYMHWFQQK PGTSPKLWIY run)STSNLASGVP ARFSGSGSGT SYSLTISRME AEDAATYYCQ QRSSYPLTFG _CD28_AGTKLELKRA AAGSGGSGII HVKGKHLCPS PLFPGPSKPF WVLVVVGGVL CD40ACYSLLVTVA FIIFWVRSKR SRLLHSDYMN MTPRRPGPTR KHYQPYAPPRDFAAYRSKKV AKKPTNKAPH PKQEPQEINF PDDLPGSNTA APVQETLHGCQPVTQEDGKE SRISVQERQ 161 hIgGk-MEAPAQLLFL LLLWLPDTTR QVQLQQSGAE LVRSGTSVKL SCTASGFNIK VIIIDSYMHWLRQG PEQGLEWIGW IDPENGDTEY APKFQGKATF TTDTSSNTAY _MFE23(K >LQLSSLTSED TAVYYCNEGT PTGPYYFDYW GQGTTVTVSS GGGGSGGGGS Q)GGGGSENVLT QSPAIMSASP GEKVTITCSA SSSVSYMHWF QQKPGTSPKL _spCD28(tWIYSTSNLAS GVPARFSGSG SGTSYSLTIS RMEAEDAATY YCQQRSSYPL run)TFGAGTKLEL KRAAAGSGGS GIIHVKGKHL CPSPLFPGPS KPFWVLVVVG _CD28_GVLACYSLLV TVAFIIFWVR SKRSRLLHSD YMNMTPRRPG PTRKHYQPYA CD40PPRDFAAYRS KKVAKKPTNK APHPKQEPQE INFPDDLPGS NTAAPVQETLHGCQPVTQED GKESRISVQE RQ 162 OSM_HuMFEMGVLLTQRTL LSLVLALLFP SMASMQVKLE QSGAEVVKPG ASVKLSCKAS 23GFNIKDSYMH WLRQGPGQRL EWIGWIDPEN GDTEYAPKFQ GKATFTTDTS _spCD28(tANTAYLGLSS LRPEDTAVYY CNEGTPTGPY YFDYWGQGTL VTVSSGGGGS run)GGGGSGGGGS ENVLTQSPSS MSASVGDRVN IACSASSSVS YMHWFQQKPG _CD28_KSPKLWIYST SNLASGVPSR FSGSGSGTDY SLTISSMQPE DAATYYCQQR CD40SSYPLTFGGG TKLEIKAAAG SGGSGIIHVK GKHLCPSPLF PGPSKPFWVL CTP244VVVGGVLACY SLLVTVAFII FWVRSKRSRL LHSDYMNMTP RRPGPTRKHYQPYAPPRDFA AYRSKKVAKK PTNKAPHPKQ EPQEINFPDD LPGSNTAAPVQETLHGCQPV TQEDGKESRI SVQERQ 163 CD8a_HuMFMALPVTALLL PLALLLHAAR PQVKLEQSGA EVVKPGASVK LSCKASGFNI E23KDSYMHWLRQ GPGQRLEWIG WIDPENGDTE YAPKFQGKAT FTTDTSANTA _spCD28(tYLGLSSLRPE DTAVYYCNEG TPTGPYYFDY WGQGTLVTVS SGGGGSGGGG run)SGGGGSENVL TQSPSSMSAS VGDRVNIACS ASSSVSYMHW FQQKPGKSPK _CD28_LWIYSTSNLA SGVPSRFSGS GSGTDYSLTI SSMQPEDAAT YYCQQRSSYP CD40LTFGGGTKLE IKAAAGSGGS GIIHVKGKHL CPSPLFPGPS KPFWVLVVVGGVLACYSLLV TVAFIIFWVR SKRSRLLHSD YMNMTPRRPG PTRKHYQPYAPPRDFAAYRS KKVAKKPTNK APHPKQEPQE INFPDDLPGS NTAAPVQETLHGCQPVTQED GKESRISVQE RQ 164 CD2_HuMFEMSFPCKFVAS FLLIFNVSSK GAVSQVKLEQ SGAEWKPGA SVKLSCKASG 23FNIKDSYMHW LRQGPGQRLE WIGWIDPENG DTEYAPKFQG KATFTTDTSA _spCD28(tNTAYLGLSSL RPEDTAVYYC NEGTPTGPYY FDYWGQGTLV TVSSGGGGSG run)GGGSGGGGSE NVLTQSPSSM SASVGDRVNI ACSASSSVSY MHWFQQKPGK _CD28_SPKLWIYSTS NLASGVPSRF SGSGSGTDYS LTISSMQPED AATYYCQQRS CD40SYPLTFGGGT KLEIKAAAGS GGSGIIHVKG KHLCPSPLFP GPSKPFWVLVVVGGVLACYS LLVTVAFIIF WVRSKRSRLL HSDYMNMTPR RPGPTRKHYQPYAPPRDFAA YRSKKVAKKP TNKAPHPKQE PQEINFPDDL PGSNTAAPVQETLHGCQPVT QEDGKESRIS VQERQ 165 IL2_HuMFEMYRMQLLSCI ALSLALVTNS QVKLEQSGAE VVKPGASVKL SCKASGFNIK 23DSYMHWLRQG PGQRLEWIGW IDPENGDTEY APKFQGKATF TTDTSANTAY _spCD28(tLGLSSLRPED TAVYYCNEGT PTGPYYFDYW GQGTLVTVSS GGGGSGGGGS run)GGGGSENVLT QSPSSMSASV GDRVNIACSA SSSVSYMHWF QQKPGKSPKL _CD28_WIYSTSNLAS GVPSRFSGSG SGTDYSLTIS SMQPEDAATY YCQQRSSYPL CD40TFGGGTKLEI KAAAGSGGSG IIHVKGKHLC PSPLFPGPSK PFWVLVVVGGVLACYSLLVT VAFIIFWVRS KRSRLLHSDY MNMTPRRPGP TRKHYQPYAPPRDFAAYRSK KVAKKPTNKA PHPKQEPQEI NFPDDLPGSN TAAPVQETLHGCQPVTQEDG KESRISVQER Q 166 GM-MWLQSLLLLG TVACSISQVK LEQSGAEVVK PGASVKLSCK ASGFNIKDSY CSF_HuMFEMHWLRQGPGQ RLEWIGWIDP ENGDTEYAPK FQGKATFTTD TSANTAYLGL 23SSLRPEDTAV YYCNEGTPTG PYYFDYWGQG TLVTVSSGGG GSGGGGSGGG _spCD28(tGSENVLTQSP SSMSASVGDR VNIACSASSS VSYMHWFQQK PGKSPKLWIY run)STSNLASGVP SRFSGSGSGT DYSLTISSMQ PEDAATYYCQ QRSSYPLTFG _CD28_GGTKLEIKAA AGSGGSGIIH VKGKHLCPSP LFPGPSKPFW VLVVVGGVLA CD40CYSLLVTVAF IIFWVRSKRS RLLHSDYMNM TPRRPGPTRK HYQPYAPPRDFAAYRSKKVA KKPTNKAPHP KQEPQEINFP DDLPGSNTAA PVQETLHGCQPVTQEDGKES RISVQERQ 167 hIgGk-MEAPAQLLFL LLLWLPDTTR QVKLEQSGAE VVKPGASVKL SCKASGFNIK VIIIDSYMHWLRQG PGQRLEWIGW IDPENGDTEY APKFQGKATF TTDTSANTAY _HuMFE23LGLSSLRPED TAVYYCNEGT PTGPYYFDYW GQGTLVTVSS GGGGSGGGGS _spCD28(tGGGGSENVLT QSPSSMSASV GDRVNIACSA SSSVSYMHWF QQKPGKSPKL run)WIYSTSNLAS GVPSRFSGSG SGTDYSLTIS SMQPEDAATY YCQQRSSYPL _CD28_TFGGGTKLEI KAAAGSGGSG IIHVKGKHLC PSPLFPGPSK PFWVLVVVGG CD40VLACYSLLVT VAFIIFWVRS KRSRLLHSDY MNMTPRRPGP TRKHYQPYAPPRDFAAYRSK KVAKKPTNKA PHPKQEPQEI NFPDDLPGSN TAAPVQETLHGCQPVTQEDG KESRISVQER Q 168 OSM_CEA6MGVLLTQRTL LSLVLALLFP SMASMQVQLV QSGAEVKKPG SSVKVSCKAS _spCD28(tGGTFSNSPIN WLRQAPGQGL EWMGSIIPSF GTANYAQKFQ GRLTITADES run)TSTAYMELSS LRSEDTAVYY CAGRSHNYEL YYYYMDVWGQ GTMVTVSSGG _CD28_GGSGGGGSGG GGSDIQMTQS PSTLSASIGD RVTITCRASE GIYHWLAWYQ CD40QKPGKAPKLL IYKASSLASG APSRFSGSGS GTDFTLTISS LQPDDFATYYCQQYSNYPLT FGGGTKLEIK RAAAGSGGSG IIHVKGKHLC PSPLFPGPSKPFWVLVVVGG VLACYSLLVT VAFIIFWVRS KRSRLLHSDY MNMTPRRPGPTRKHYQPYAP PRDFAAYRSK KVAKKPTNKA PHPKQEPQEI NFPDDLPGSNTAAPVQETLH GCQPVTQEDG KESRISVQER Q 169 CD8a_CEA6MALPVTALLL PLALLLHAAR PQVQLVQSGA EVKKPGSSVK VSCKASGGTF _spCD28(tSNSPINWLRQ APGQGLEWMG SIIPSFGTAN YAQKFQGRLT ITADESTSTA run)YMELSSLRSE DTAVYYCAGR SHNYELYYYY MDVWGQGTMV TVSSGGGGSG _CD28_GGGSGGGGSD IQMTQSPSTL SASIGDRVTI TGRASEGIYH WLAWYQQKPG CD40KAPKLLIYKA SSLASGAPSR FSGSGSGTDF TLTISSLQPD DFATYYCQQYSNYPLTFGGG TKLEIKRAAA GSGGSGIIHV KGKHLCPSPL FPGPSKPFWVLVVVGGVLAC YSLLVTVAFI IFWVRSKRSR LLHSDYMNMT PRRPGPTRKHYQPYAPPRDF AAYRSKKVAK KPTNKAPHPK QEPQEINFPD DLPGSNTAAPVQETLHGCQP VTQEDGKESR ISVQERQ 170 CD2_CEA6MSFPCKFVAS FLLIFNVSSK GAVSQVQLVQ SGAEVKKPGS SVKVSCKASG _spCD28(tGTFSNSPINW LRQAPGQGLE WMGSIIPSFG TANYAQKFQG RLTITADEST run)STAYMELSSL RSEDTAVYYC AGRSHNYELY YYYMDVWGQG TMVTVSSGGG _CD28_GSGGGGSGGG GSDIQMTQSP STLSASIGDR VTITCRASEG IYHWLAWYQQ CD40KPGKAPKLLI YKASSLASGA PSRFSGSGSG TDFTLTISSL QPDDFATYYCQQYSNYPLTF GGGTKLEIKR AAAGSGGSGI IHVKGKHLCP SPLFPGPSKPFWVLVVVGGV LACYSLLVTV AFIIFWVRSK RSRLLHSDYM NMTPRRPGPTRKHYQPYAPP RDFAAYRSKK VAKKPTNKAP HPKQEPQEIN FPDDLPGSNTAAPVQETLHG CQPVTQEDGK ESRISVQERQ 171 IL2_CEA6MYRMQLLSCI ALSLALVTNS QVQLVQSGAE VKKPGSSVKV SCKASGGTFS _spCD28(tNSPINWLRQA PGQGLEWMGS IIPSFGTANY AQKFQGRLTI TADESTSTAY run)MELSSLRSED TAVYYCAGRS HNYELYYYYM DVWGQGTMVT VSSGGGGSGG _CD28_GGSGGGGSDI QMTQSPSTLS ASIGDRVTIT CRASEGIYHW LAWYQQKPGK CD40APKLLIYKAS SLASGAPSRF SGSGSGTDFT LTISSLQPDD FATYYCQQYSNYPLTFGGGT KLEIKRAAAG SGGSGIIHVK GKHLCPSPLF PGPSKPFWVLVVVGGVLACY SLLVTVAFII FWVRSKRSRL LHSDYMNMTP RRPGPTRKHYQPYAPPRDFA AYRSKKVAKK PTNKAPHPKQ EPQEINFPDD LPGSNTAAPVQETLHGCQPV TQEDGKESRI SVQERQ 172 GM-MWLQSLLLLG TVACSISQVQ LVQSGAEVKK PGSSVKVSCK ASGGTFSNSP CSF_CEA6INWLRQAPGQ GLEWMGSIIP SFGTANYAQK FQGRLTITAD ESTSTAYMEL _spCD28(tSSLRSEDTAV YYCAGRSHNY ELYYYYMDVW GQGTMVTVSS GGGGSGGGGS run)GGGGSDIQMT QSPSTLSASI GDRVTITCRA SEGIYHWLAW YQQKPGKAPK _CD28_LLIYKASSLA SGAPSRFSGS GSGTDFTLTI SSLQPDDFAT YYCQQYSNYP CD40LTFGGGTKLE IKRAAAGSGG SGIIHVKGKH LCPSPLFPGP SKPFWVLVVVGGVLACYSLL VTVAFIIFWV RSKRSRLLHS DYMNMTPRRP GPTRKHYQPYAPPRDFAAYR SKKVAKKPTN KAPHPKQEPQ EINFPDDLPG SNTAAPVQETLHGCQPVTQE DGKESRISVQ ERQ 173 hIgGk-MEAPAQLLFL LLLWLPDTTR QVQLVQSGAE VKKPGSSVKV SCKASGGTFS VIIINSPINWLRQA PGQGLEWMGS IIPSFGTANY AQKFQGRLTI TADESTSTAY _CEA6MELSSLRSED TAVYYCAGRS HNYELYYYYM DVWGQGTMVT VSSGGGGSGG _spCD28(tGGSGGGGSDI QMTQSPSTLS ASIGDRVTIT CRASEGIYHW LAWYQQKPGK run)APKLLIYKAS SLASGAPSRF SGSGSGTDFT LTISSLQPDD FATYYCQQYS _CD28_NYPLTFGGGT KLEIKRAAAG SGGSGIIHVK GKHLCPSPLF PGPSKPFWVL CD40VVVGGVLACY SLLVTVAFII FWVRSKRSRL LHSDYMNMTP RRPGPTRKHYQPYAPPRDFA AYRSKKVAKK PTNKAPHPKQ EPQEINFPDD LPGSNTAAPVQETLHGCQPV TQEDGKESRI SVQERQ 174 OSM_BW431MGVLLTQRTL LSLVLALLFP SMASMQLQES GPGLVRPSQT LSLTCTVSGF /26TISSGYSWHW VRQPPGRGLE WIGYIQYSGI TNYNPSLKSR VTMLVDTSKN _spCD28(tQFSLRLSSVT AADTAVYYCA REDYDYHWYF DVWGQGSLVT VSSGGGGSGG run)GGSGGGGSGV HSDIQMTQSP SSLSASVGDR VTITCSTSSS VSYMHWYQQK _CD28_PGKAPKLLIY STSNLASGVP SRFSGSGSGT DFTFTISSLQ PEDIATYYCH CD40QWSSYPTFGQ GTKVEIKRAA AGSGGSGIIH VKGKHLCPSP LFPGPSKPFWVLVVVGGVLA CYSLLVTVAF IIFWVRSKRS RLLHSDYMNM TPRRPGPTRKHYQPYAPPRD FAAYRSKKVA KKPTNKAPHP KQEPQEINFP DDLPGSNTAAPVQETLHGCQ PVTQEDGKES RISVQERQ 175 CD8a_BW43MALPVTALLL PLALLLHAAR PQLQESGPGL VRPSQTLSLT CTVSGFTISS l/26_spCDGYSWHWVRQP PGRGLEWIGY IQYSGITNYN PSLKSRVTML VDTSKNQFSL 28(trun)RLSSVTAADT AVYYCAREDY DYHWYFDVWG QGSLVTVSSG GGGSGGGGSG _CD28_GGGSGVHSDI QMTQSPSSLS ASVGDRVTIT CSTSSSVSYM HWYQQKPGKA CD40PKLLIYSTSN LASGVPSRFS GSGSGTDFTF TISSLQPEDI ATYYCHQWSSYPTFGQGTKV EIKRAAAGSG GSGIIHVKGK HLCPSPLFPG PSKPFWVLVVVGGVLACYSL LVTVAFIIFW VRSKRSRLLH SDYMNMTPRR PGPTRKHYQPYAPPRDFAAY RSKKVAKKPT NKAPHPKQEP QEINFPDDLP GSNTAAPVQETLHGCQPVTQ EDGKESRISV QERQ 176 CD2_BW431MSFPCKFVAS FLLIFNVSSK GAVSQLQESG PGLVRPSQTL SLTCTVSGFT /26ISSGYSWHWV RQPPGRGLEW IGYIQYSGIT NYNPSLKSRV TMLVDTSKNQ _spCD28(tFSLRLSSVTA ADTAVYYCAR EDYDYHWYFD VWGQGSLVTV SSGGGGSGGG run)GSGGGGSGVH SDIQMTQSPS SLSASVGDRV TITCSTSSSV SYMHWYQQKP _CD28_GKAPKLLIYS TSNLASGVPS RFSGSGSGTD FTFTISSLQP EDIATYYCHQ CD40WSSYPTFGQG TKVEIKRAAA GSGGSGIIHV KGKHLCPSPL FPGPSKPFWVLVVVGGVLAC YSLLVTVAFI IFWVRSKRSR LLHSDYMNMT PRRPGPTRKHYQPYAPPRDF AAYRSKKVAK KPTNKAPHPK QEPQEINFPD DLPGSNTAAPVQETLHGCQP VTQEDGKESR ISVQERQ 177 IL2_BW431MYRMQLLSCI ALSLALVTNS QLQESGPGLV RPSQTLSLTC TVSGFTISSG /26YSWHWVRQPP GRGLEWIGYI QYSGITNYNP SLKSRVTMLV DTSKNQFSLR _spCD28(tLSSVTAADTA VYYCAREDYD YHWYFDVWGQ GSLVTVSSGG GGSGGGGSGG run)GGSGVHSDIQ MTQSPSSLSA SVGDRVTITC STSSSVSYMH WYQQKPGKAP _CD28_KLLIYSTSNL ASGVPSRFSG SGSGTDFTFT ISSLQPEDIA TYYCHQWSSY CD40PTFGQGTKVE IKRAAAGSGG SGIIHVKGKH LCPSPLFPGP SKPFWVLVVVGGVLACYSLL VTVAFIIFWV RSKRSRLLHS DYMNMTPRRP GPTRKHYQPYAPPRDFAAYR SKKVAKKPTN KAPHPKQEPQ EINFPDDLPG SNTAAPVQETLHGCQPVTQE DGKESRISVQ ERQ 178 GM-CSFMWLQSLLLLG TVACSISQLQ ESGPGLVRPS QTLSLTCTVS GFTISSGYSW _BW431/26HWVRQPPGRG LEWIGYIQYS GITNYNPSLK SRVTMLVDTS KNQFSLRLSS _spCD28(tVTAADTAVYY CAREDYDYHW YFDVWGQGSL VTVSSGGGGS GGGGSGGGGS run)GVHSDIQMTQ SPSSLSASVG DRVTITCSTS SSVSYMHWYQ QKPGKAPKLL _CD28_IYSTSNLASG VPSRFSGSGS GTDFTFTISS LQPEDIATYY CHQWSSYPTF CD40GQGTKVEIKR AAAGSGGSGI IHVKGKHLCP SPLFPGPSKP FWVLVVVGGVLACYSLLVTV AFIIFWVRSK RSRLLHSDYM NMTPRRPGPT RKHYQPYAPPRDFAAYRSKK VAKKPTNKAP HPKQEPQEIN FPDDLPGSNT AAPVQETLHGCQPVTQEDGK ESRISVQERQ 179 hIgGk-MEAPAQLLFL LLLWLPDTTR QLQESGPGLV RPSQTLSLTC TVSGFTISSG VIIIYSWHWVRQPP GRGLEWIGYI QYSGITNYNP SLKSRVTMLV DTSKNQFSLR _BW431/26LSSVTAADTA VYYCAREDYD YHWYFDVWGQ GSLVTVSSGG GGSGGGGSGG _spCD28(tGGSGVHSDIQ MTQSPSSLSA SVGDRVTITC STSSSVSYMH WYQQKPGKAP run)KLLIYSTSNL ASGVPSRFSG SGSGTDFTFT ISSLQPEDIA TYYCHQWSSY _CD28_PTFGQGTKVE IKRAAAGSGG SGIIHVKGKH LCPSPLFPGP SKPFWVLVVV CD40GGVLACYSLL VTVAFIIFWV RSKRSRLLHS DYMNMTPRRP GPTRKHYQPYAPPRDFAAYR SKKVAKKPTN KAPHPKQEPQ EINFPDDLPG SNTAAPVQETLHGCQPVTQE DGKESRISVQ ERQ 180 OSM_HuT84.MGVLLTQRTL LSLVLALLFP SMASMEVQLV ESGGGLVQPG GSLRLSCAAS 66GFNIKDTYMH WVRQAPGKGL EWVARIDPAN GNSKYADSVK GRFTISADTS _spCD28(tKNTAYLQMNS LRAEDTAVYY CAPFGYYVSD YAMAYWGQGT LVTVSSGGGG run)SGGGGSGGGG SDIQLTQSPS SLSASVGDRV TITCRAGESV DIFGVGFLHW _CD28_YQQKPGKAPK LLIYRASNLE SGVPSRFSGS GSRTDFTLTI SSLQPEDFAT CD40YYCQQTNEDP YTFGQGTKVE IKAAAGSGGS GIIHVKGKHL CPSPLFPGPSKPFWVLVVVG GVLACYSLLV TVAFIIFWVR SKRSRLLHSD YMNMTPRRPGPTRKHYQPYA PPRDFAAYRS KKVAKKPTNK APHPKQEPQE INFPDDLPGSNTAAPVQETL HGCQPVTQED GKESRISVQE RQ 181 CD8a_HuT8MALPVTALLL PLALLLHAAR PEVQLVESGG GLVQPGGSLR LSCAASGFNI 4.66KDTYMHWVRQ APGKGLEWVA RIDPANGNSK YADSVKGRFT ISADTSKNTA _spCD28(tYLQMNSLRAE DTAVYYCAPF GYYVSDYAMA YWGQGTLVTV SSGGGGSGGG run)GSGGGGSDIQ LTQSPSSLSA SVGDRVTITC RAGESVDIFG VGFLHWYQQK _CD28_PGKAPKLLIY RASNLESGVP SRFSGSGSRT DFTLTISSLQ PEDFATYYCQ CD40QTNEDPYTFG QGTKVEIKAA AGSGGSGIIH VKGKHLCPSP LFPGPSKPFWVLVVVGGVLA CYSLLVTVAF IIFWVRSKRS RLLHSDYMNM TPRRPGPTRKHYQPYAPPRD FAAYRSKKVA KKPTNKAPHP KQEPQEINFP DDLPGSNTAAPVQETLHGCQ PVTQEDGKES RISVQERQ 182 CD2_HuT84.MSFPCKFVAS FLLIFNVSSK GAVSEVQLVE SGGGLVQPGG SLRLSCAASG 66FNIKDTYMHW VRQAPGKGLE WVARIDPANG NSKYADSVKG RFTISADTSK _spCD28(tNTAYLQMNSL RAEDTAVYYC APFGYYVSDY AMAYWGQGTL VTVSSGGGGS run)GGGGSGGGGS DIQLTQSPSS LSASVGDRVT ITCRAGESVD IFGVGFLHWY _CD28_QQKPGKAPKL LIYRASNLES GVPSRFSGSG SRTDFTLTIS SLQPEDFATY CD40YCQQTNEDPY TFGQGTKVEI KAAAGSGGSG IIHVKGKHLC PSPLFPGPSKPFWVLVVVGG VLACYSLLVT VAFIIFWVRS KRSRLLHSDY MNMTPRRPGPTRKHYQPYAP PRDFAAYRSK KVAKKPTNKA PHPKQEPQEI NFPDDLPGSNTAAPVQETLH GCQPVTQEDG KESRISVQER Q 183 IL2_HuT84MYRMQLLSCI ALSLALVTNS EVQLVESGGG LVQPGGSLRL SCAASGFNIK . 66DTYMHWVRQA PGKGLEWVAR IDPANGNSKY ADSVKGRFTI SADTSKNTAY _spCD28(tLQMNSLRAED TAVYYCAPFG YYVSDYAMAY WGQGTLVTVS SGGGGSGGGG run)SGGGGSDIQL TQSPSSLSAS VGDRVTITCR AGESVDIFGV GFLHWYQQKP _CD28_GKAPKLLIYR ASNLESGVPS RFSGSGSRTD FTLTISSLQP EDFATYYCQQ CD40TNEDPYTFGQ GTKVEIKAAA GSGGSGIIHV KGKHLCPSPL FPGPSKPFWVLVVVGGVLAC YSLLVTVAFI IFWVRSKRSR LLHSDYMNMT PRRPGPTRKHYQPYAPPRDF AAYRSKKVAK KPTNKAPHPK QEPQEINFPD DLPGSNTAAPVQETLHGCQP VTQEDGKESR ISVQERQ 184 GM-MWLQSLLLLG TVACSISEVQ LVESGGGLVQ PGGSLRLSCA ASGFNIKDTY CSF_HuT84MHWVRQAPGK GLEWVARIDP ANGNSKYADS VKGRFTISAD TSKNTAYLQM . 66NSLRAEDTAV YYCAPFGYYV SDYAMAYWGQ GTLVTVSSGG GGSGGGGSGG _spCD28(tGGSDIQLTQS PSSLSASVGD RVTITCRAGE SVDIFGVGFL HWYQQKPGKA run)PKLLIYRASN LESGVPSRFS GSGSRTDFTL TISSLQPEDF ATYYCQQTNE _CD28_DPYTFGQGTK VEIKAAAGSG GSGIIHVKGK HLCPSPLFPG PSKPFWVLVV CD40VGGVLACYSL LVTVAFIIFW VRSKRSRLLH SDYMNMTPRR PGPTRKHYQPYAPPRDFAAY RSKKVAKKPT NKAPHPKQEP QEINFPDDLP GSNTAAPVQETLHGCQPVTQ EDGKESRISV QERQ 185 hIgGk-MEAPAQLLFL LLLWLPDTTR EVQLVESGGG LVQPGGSLRL SCAASGFNIK VIII_HuT8DTYMHWVRQA PGKGLEWVAR IDPANGNSKY ADSVKGRFTI SADTSKNTAY 4.66LQMNSLRAED TAVYYCAPFG YYVSDYAMAY WGQGTLVTVS SGGGGSGGGG _spCD28(tSGGGGSDIQL TQSPSSLSAS VGDRVTITCR AGESVDIFGV GFLHWYQQKP run)GKAPKLLIYR ASNLESGVPS RFSGSGSRTD FTLTISSLQP EDFATYYCQQ _CD28_TNEDPYTFGQ GTKVEIKAAA GSGGSGIIHV KGKHLCPSPL FPGPSKPFWV CD40LVVVGGVLAC YSLLVTVAFI IFWVRSKRSR LLHSDYMNMT PRRPGPTRKHYQPYAPPRDF AAYRSKKVAK KPTNKAPHPK QEPQEINFPD DLPGSNTAAPVQETLHGCQP VTQEDGKESR ISVQERQ 186 SS1QVQLQQSGPE LEKPGASVKL SCKASGYSFT GYTMNWVKQS HGKSLEWIGLITPYNGASSY NQKFRGKATL TVDKSSSTAY MDLLSLTSED SAVYFCARGGYDGRGFDYWG QGTTVTVSSG GGGSGGGGSG GGGSDIELTQ SPAIMSASPGEKVTMTCSAS SSVSYMHWYQ QKSGTSPKRW IYDTSKLASG VPGRFSGSGSGNSYSLTISS VEAEDDATYY CQQWSKHPLT FGAGTKLEIK 187 M5QVQLVQSGAE VEKPGASVKV SCKASGYTFT DYYMHWVRQA PGQGLEWMGW (humanisedINPNSGGTNY AQKFQGRVTM TRDTSISTAY MELSRLRSDD TAVYYCASGW SS1)DFDYWGQGTL VTVSSGGGGS GGGGSGGGGS DIVMTQSPSS LSASVGDRVTITCRASQSIR YYLSWYQQKP GKAPKLLIYT ASILQNGVPS RFSGSGSGTDFTLTISSLQP EDFATYYCLQ TYTTPDFGPG TKVEIK 188 HN1QVQLVQSGAE VKRPGASVQV SCRASGYSIN TYYMQWVRQA PGAGLEWMGVINPSGVTSYA QKFQGRVTLT NDTSTNTVYM QLNSLTSADT AVYYCARWALWGDFGMDVWG KGTLVTVSSG GGGSGGGGSG GGGSDIQMTQ SPSTLSASIGDRVTITCRAS EGIYHWLAWY QQKPGKAPKL LIYKASSLAS GAPSRFSGSGSGTDFTLTIS SLQPDDFATY YCQQYSNYPL TFGGGTKLEI K 189 M912QVQLQESGPG LVKPSETLSL TCTVSGGSVS SGSYYWSWIR QPPGKGLEWIGYIYYSGSTN YNPSLKSRVT ISVDTSKNQF SLKLSSVTAA DTAVYYCAREGKNGAFDIWG QGTMVTVSSG GGGSGGGGSG GGGSDIQMTQ SPSSLSASVGDRVTITCRAS QSISSYLNWY QQKPGKAPKL LIYAASSLQS GVPSGFSGSGSGTDFTLTIS SLQPEDFATY YCQQSYSTPL TFGGGTKVEI K 190 HuYP218EVQLVESGGG LVQPGGSLRL SCAASGFDLG FYFYACWVRQ APGKGLEWVSGIYTAGSGST YYASWAKGRF TISRDNSKNT LYLQMNSLRA EDTAVYYCARSTANTRSTYY LNLWGQGTLV TVSSGGGGSG GGGSGGGGSD IQMTQSPSSLSASVGDRVTI TCQASQRISS YLSWYQQKPG KVPKLLIYGA STLASGVPSRFSGSGSGTDF TLTISSLQPE DVATYYCQSY AYFDSNNWHA FGGGTKVEI 191 P4QVQLQQSGPG LVTPSQTLSL TCAISGDSVS SNSATWNWIR QSPSRGLEWLGRTYYRSKWY NDYAVSVKSR MSINPDTSKN QFSLQLNSVT PEDTAVYYCARGMMTYYYGM dvwgqgttvt vssggggsgg ggsggggsqp vltqssslsaSPGASASLTC TLRSGINVGP YRIYWYQQKP GSPPQYLLNY KSDSDKQQGSGVPSRFSGSK DASANAGVLL ISGLRSEDEA DYYCMIWHSS AAVFGGGTQL TVLS 192 OSMSSMGVLLTQRTL LSLVLALLFP SMASMQVOLQ QSGPELEKPG ASVKLSCKAS _spCD28GYSFTGYTMN WVKQSHGKSL EWIGLITPYN GASSYNQKFR GKATLTVDKS _CD28_SSTAYMDLLS LTSEDSAVYF CARGGYDGRG FDYWGQGTTV TVSSGGGGSG CD40GGGSGGGGSD IELTQSPAIM SASPGEKVTM TCSASSSVSY MHWYQQKSGT CTP224SPKRWIYDTS KLASGVPGRF SGSGSGNSYS LTISSVEAED DATYYCQQWSKHPLTFGAGT KLEIKAAAGS GGSGILVKQS PMLVAYDNAV NLSCKYSYNLFSREFRASLH KGLDSAVEVC VVYGNYSQQL QVYSKTGFNC DGKLGNESVTFYLQNLYVNQ TDIYFCKIEV MYPPPYLDNE KSNGTIIHVK GKHLCPSPLFPGPSKPFWVL VVVGGVLACY SLLVTVAFII FWVRSKRSRL LHSDYMNMTPRRPGPTRKHY QPYAPPRDFA AYRSKKVAKK PTNKAPHPKQ EPQEINFPDDLPGSNTAAPV QETLHGCQPV TQEDGKESRI SVQERQ 193 CD8a_SS1MALPVTALLL PLALLLHAAR PQVQLQQSGP ELEKPGASVK LSCKASGYSF _spCD28TGYTMNWVKQ SHGKSLEWIG LITPYNGASS YNQKFRGKAT LTVDKSSSTA _CD28_YMDLLSLTSE DSAVYFCARG GYDGRGFDYW GQGTTVTVSS GGGGSGGGGS CD40GGGGSDIELT QSPAIMSASP GEKVTMTCSA SSSVSYMHWY QQKSGTSPKRWIYDTSKLAS GVPGRFSGSG SGNSYSLTIS SVEAEDDATY YCQQWSKHPLTFGAGTKLEI KAAAGSGGSG ILVKQSPMLV AYDNAVNLSC KYSYNLFSREFRASLHKGLD SAVEVCVVYG NYSQQLQVYS KTGFNCDGKL GNESVTFYLQNLYVNQTDIY FCKIEVMYPP PYLDNEKSNG TIIHVKGKHL CPSPLFPGPSKPFWVLVVVG GVLACYSLLV TVAFIIFWVR SKRSRLLHSD YMNMTPRRPGPTRKHYQPYA PPRDFAAYRS KKVAKKPTNK APHPKQEPQE INFPDDLPGSNTAAPVQETL HGCQPVTQED GKESRISVQE RQ 194 CD2_SS1MSFPCKFVAS FLLIFNVSSK GAVSQVQLQQ SGPELEKPGA SVKLSCKASG _spCD28YSFTGYTMNW VKQSHGKSLE WIGLITPYNG ASSYNQKFRG KATLTVDKSS _CD28_STAYMDLLSL TSEDSAVYFC ARGGYDGRGF DYWGQGTTVT VSSGGGGSGG CD40GGSGGGGSDI ELTQSPAIMS ASPGEKVTMT CSASSSVSYM HWYQQKSGTSPKRWIYDTSK LASGVPGRFS GSGSGNSYSL TISSVEAEDD ATYYCQQWSKHPLTFGAGTK LEIKAAAGSG GSGILVKQSP MLVAYDNAVN LSCKYSYNLFSREFRASLHK GLDSAVEVCV VYGNYSQQLQ VYSKTGFNCD GKLGNESVTFYLQNLYVNQT DIYFCKIEVM YPPPYLDNEK SNGTIIHVKG KHLCPSPLFPGPSKPFWVLV VVGGVLACYS LLVTVAFIIF WVRSKRSRLL HSDYMNMTPRRPGPTRKHYQ PYAPPRDFAA YRSKKVAKKP TNKAPHPKQE PQEINFPDDLPGSNTAAPVQ ETLHGCQPVT QEDGKESRIS VQERQ 195 IL2_SS1MYRMQLLSCI ALSLALVTNS QVQLQQSGPE LEKPGASVKL SCKASGYSFT _spCD28GYTMNWVKQS HGKSLEWIGL ITPYNGASSY NQKFRGKATL TVDKSSSTAY _CD28_MDLLSLTSED SAVYFCARGG YDGRGFDYWG QGTTVTVSSG GGGSGGGGSG CD40GGGSDIELTQ SPAIMSASPG EKVTMTCSAS SSVSYMHWYQ QKSGTSPKRWIYDTSKLASG VPGRFSGSGS GNSYSLTISS VEAEDDATYY CQQWSKHPLTFGAGTKLEIK AAAGSGGSGI LVKQSPMLVA YDNAVNLSCK YSYNLFSREFRASLHKGLDS AVEVCVVYGN YSQQLQVYSK TGFNCDGKLG NESVTFYLQNLYVNQTDIYF CKIEVMYPPP YLDNEKSNGT IIHVKGKHLC PSPLFPGPSKPFWVLVVVGG VLACYSLLVT VAFIIFWVRS KRSRLLHSDY MNMTPRRPGPTRKHYQPYAP PRDFAAYRSK KVAKKPTNKA PHPKQEPQEI NFPDDLPGSNTAAPVQETLH GCQPVTQEDG KESRISVQER Q 196 GM-MWLQSLLLLG TVACSISQVQ LQQSGPELEK PGASVKLSCK ASGYSFTGYT CSF_SS1MNWVKQSHGK SLEWIGLITP YNGASSYNQK FRGKATLTVD KSSSTAYMDL _spCD28LSLTSEDSAV YFCARGGYDG RGFDYWGQGT TVTVSSGGGG SGGGGSGGGG _CD28_SDIELTQSPA IMSASPGEKV TMTCSASSSV SYMHWYQQKS GTSPKRWIYD CD40TSKLASGVPG RFSGSGSGNS YSLTISSVEA EDDATYYCQQ WSKHPLTFGAGTKLEIKAAA GSGGSGILVK QSPMLVAYDN AVNLSCKYSY NLFSREFRASLHKGLDSAVE VCVVYGNYSQ QLQVYSKTGF NCDGKLGNES VTFYLQNLYVNQTDIYFCKI EVMYPPPYLD NEKSNGTIIH VKGKHLCPSP LFPGPSKPFWVLVVVGGVLA CYSLLVTVAF IIFWVRSKRS RLLHSDYMNM TPRRPGPTRKHYQPYAPPRD FAAYRSKKVA KKPTNKAPHP KQEPQEINFP DDLPGSNTAAPVQETLHGCQ PVTQEDGKES RISVQERQ 197 hIgGk-MEAPAQLLFL LLLWLPDTTR QVQLQQSGPE LEKPGASVKL SCKASGYSFT VIII_SS1GYTMNWVKQS HGKSLEWIGL ITPYNGASSY NQKFRGKATL TVDKSSSTAY _spCD28MDLLSLTSED SAVYFCARGG YDGRGFDYWG QGTTVTVSSG GGGSGGGGSG _CD28_GGGSDIELTQ SPAIMSASPG EKVTMTCSAS SSVSYMHWYQ QKSGTSPKRW CD40IYDTSKLASG VPGRFSGSGS GNSYSLTISS VEAEDDATYY CQQWSKHPLTFGAGTKLEIK AAAGSGGSGI LVKQSPMLVA YDNAVNLSCK YSYNLFSREFRASLHKGLDS AVEVCVVYGN YSQQLQVYSK TGFNCDGKLG NESVTFYLQNLYVNQTDIYF CKIEVMYPPP YLDNEKSNGT IIHVKGKHLC PSPLFPGPSKPFWVLVVVGG VLACYSLLVT VAFIIFWVRS KRSRLLHSDY MNMTPRRPGPTRKHYQPYAP PRDFAAYRSK KVAKKPTNKA PHPKQEPQEI NFPDDLPGSNTAAPVQETLH GCQPVTQEDG KESRISVQER Q 198 OSM_SS1_sMGVLLTQRTL LSLVLALLFP SMASMQVOLQ QSGPELEKPG ASVKLSCKAS pCD8GYSFTGYTMN WVKQSHGKSL EWIGLITPYN GASSYNQKFR GKATLTVDKS _CD28_SSTAYMDLLS LTSEDSAVYF CARGGYDGRG FDYWGQGTTV TVSSGGGGSG CD40GGGSGGGGSD IELTQSPAIM SASPGEKVTM TCSASSSVSY MHWYQQKSGT CTP236SPKRWIYDTS KLASGVPGRF SGSGSGNSYS LTISSVEAED DATYYCQQWSKHPLTFGAGT KLEIKAAAGS GGSGFVPVFL PAKPTTTPAP RPPTPAPTIASQPLSLRPEA CRPAAGGAVH TRGLDFACDI YIWAPLAGTC GVLLLSLVITLYCNHRNRSK RSRLLHSDYM NMTPRRPGPT RKHYQPYAPP RDFAAYRSKKVAKKPTNKAP HPKQEPQEIN FPDDLPGSNT AAPVQETLHG CQPVTQEDGK ESRISVQERQ 199CD8a_SS1 MALPVTALLL PLALLLHAAR PQVQLQQSGP ELEKPGASVK LSCKASGYSF _spCD8TGYTMNWVKQ SHGKSLEWIG LITPYNGASS YNQKFRGKAT LTVDKSSSTA _CD28_YMDLLSLTSE DSAVYFCARG GYDGRGFDYW GQGTTVTVSS GGGGSGGGGS CD40GGGGSDIELT QSPAIMSASP GEKVTMTCSA SSSVSYMHWY QQKSGTSPKRWIYDTSKLAS GVPGRFSGSG SGNSYSLTIS SVEAEDDATY YCQQWSKHPLTFGAGTKLEI KAAAGSGGSG FVPVFLPAKP TTTPAPRPPT PAPTIASQPLSLRPEACRPA AGGAVHTRGL DFACDIYIWA PLAGTCGVLL LSLVITLYCNHRNRSKRSRL LHSDYMNMTP RRPGPTRKHY QPYAPPRDFA AYRSKKVAKKPTNKAPHPKQ EPQEINFPDD LPGSNTAAPV QETLHGCQPV TQEDGKESRI SVQERQ 200CD2_SS1_s MSFPCKFVAS FLLIFNVSSK GAVSQVQLQQ SGPELEKPGA SVKLSCKASG pCD8YSFTGYTMNW VKQSHGKSLE WIGLITPYNG ASSYNQKFRG KATLTVDKSS _CD28_STAYMDLLSL TSEDSAVYFC ARGGYDGRGF DYWGQGTTVT VSSGGGGSGG CD40GGSGGGGSDI ELTQSPAIMS ASPGEKVTMT CSASSSVSYM HWYQQKSGTSPKRWIYDTSK LASGVPGRFS GSGSGNSYSL TISSVEAEDD ATYYCQQWSKHPLTFGAGTK LEIKAAAGSG GSGFVPVFLP AKPTTTPAPR PPTPAPTIASQPLSLRPEAC RPAAGGAVHT RGLDFACDIY IWAPLAGTCG VLLLSLVITLYCNHRNRSKR SRLLHSDYMN MTPRRPGPTR KHYQPYAPPR DFAAYRSKKVAKKPTNKAPH PKQEPQEINF PDDLPGSNTA APVQETLHGC QPVTQEDGKE SRISVQERQ 201IL2_SS1_s MYRMQLLSCI ALSLALVTNS QVQLQQSGPE LEKPGASVKL SCKASGYSFT pCD8GYTMNWVKQS HGKSLEWIGL ITPYNGASSY NQKFRGKATL TVDKSSSTAY _CD28_MDLLSLTSED SAVYFCARGG YDGRGFDYWG QGTTVTVSSG GGGSGGGGSG CD40GGGSDIELTQ SPAIMSASPG EKVTMTCSAS SSVSYMHWYQ QKSGTSPKRWIYDTSKLASG VPGRFSGSGS GNSYSLTISS VEAEDDATYY CQQWSKHPLTFGAGTKLEIK AAAGSGGSGF VPVFLPAKPT TTPAPRPPTP APTIASQPLSLRPEACRPAA GGAVHTRGLD FACDIYIWAP LAGTCGVLLL SLVITLYCNHRNRSKRSRLL HSDYMNMTPR RPGPTRKHYQ PYAPPRDFAA YRSKKVAKKPTNKAPHPKQE PQEINFPDDL PGSNTAAPVQ ETLHGCQPVT QEDGKESRIS VQERQ 202 GM-MWLQSLLLLG TVACSISQVQ LQQSGPELEK PGASVKLSCK ASGYSFTGYT CSF_SS1MNWVKQSHGK SLEWIGLITP YNGASSYNQK FRGKATLTVD KSSSTAYMDL _spCD8LSLTSEDSAV YFCARGGYDG RGFDYWGQGT TVTVSSGGGG SGGGGSGGGG _CD28_SDIELTQSPA IMSASPGEKV TMTCSASSSV SYMHWYQQKS GTSPKRWIYD CD40TSKLASGVPG RFSGSGSGNS YSLTISSVEA EDDATYYCQQ WSKHPLTFGAGTKLEIKAAA GSGGSGFVPV FLPAKPTTTP APRPPTPAPT IASQPLSLRPEACRPAAGGA VHTRGLDFAC DIYIWAPLAG TCGVLLLSLV ITLYCNHRNRSKRSRLLHSD YMNMTPRRPG PTRKHYQPYA PPRDFAAYRS KKVAKKPTNKAPHPKQEPQE INFPDDLPGS NTAAPVQETL HGCQPVTQED GKESRISVQE RQ 203 hIgGk-MEAPAQLLFL LLLWLPDTTR QVQLQQSGPE LEKPGASVKL SCKASGYSFT VIIIGYTMNWVKQS HGKSLEWIGL ITPYNGASSY NQKFRGKATL TVDKSSSTAY _SS1_spCDMDLLSLTSED SAVYFCARGG YDGRGFDYWG QGTTVTVSSG GGGSGGGGSG 8GGGSDIELTQ SPAIMSASPG EKVTMTCSAS SSVSYMHWYQ QKSGTSPKRW _CD28_IYDTSKLASG VPGRFSGSGS GNSYSLTISS VEAEDDATYY CQQWSKHPLT CD40FGAGTKLEIK AAAGSGGSGF VPVFLPAKPT TTPAPRPPTP APTIASQPLSLRPEACRPAA GGAVHTRGLD FACDIYIWAP LAGTCGVLLL SLVITLYCNHRNRSKRSRLL HSDYMNMTPR RPGPTRKHYQ PYAPPRDFAA YRSKKVAKKPTNKAPHPKQE PQEINFPDDL PGSNTAAPVQ ETLHGCQPVT QEDGKESRIS VQERQ 204 OSM_SS1MGVLLTQRTL LSLVLALLFP SMASMQVQLQ QSGPELEKPG ASVKLSCKAS _CD28TMGYSFTGYTMN WVKQSHGKSL EWIGLITPYN GASSYNQKFR GKATLTVDKS _CD28_SSTAYMDLLS LTSEDSAVYF CARGGYDGRG FDYWGQGTTV TVSSGGGGSG CD40GGGSGGGGSD IELTQSPAIM SASPGEKVTM TCSASSSVSY MHWYQQKSGTSPKRWIYDTS KLASGVPGRF SGSGSGNSYS LTISSVEAED DATYYCQQWSKHPLTFGAGT KLEIKAAAGS GGSGFWVLVV VGGVLACYSL LVTVAFIIFWVRSKRSRLLH SDYMNMTPRR PGPTRKHYQP YAPPRDFAAY RSKKVAKKPTNKAPHPKQEP QEINFPDDLP GSNTAAPVQE TLHGCQPVTQ EDGKESRISV QERQ 205 CD8a_SS1MALPVTALLL PLALLLHAAR PQVQLQQSGP ELEKPGASVK LSCKASGYSF _CD28TMTGYTMNWVKQ SHGKSLEWIG LITPYNGASS YNQKFRGKAT LTVDKSSSTA _CD28_YMDLLSLTSE DSAVYFCARG GYDGRGFDYW GQGTTVTVSS GGGGSGGGGS CD40GGGGSDIELT QSPAIMSASP GEKVTMTCSA SSSVSYMHWY QQKSGTSPKRWIYDTSKLAS GVPGRFSGSG SGNSYSLTIS SVEAEDDATY YCQQWSKHPLTFGAGTKLEI KAAAGSGGSG FWVLVVVGGV LACYSLLVTV AFIIFWVRSKRSRLLHSDYM NMTPRRPGPT RKHYQPYAPP RDFAAYRSKK VAKKPTNKAPHPKQEPQEIN FPDDLPGSNT AAPVQETLHG CQPVTQEDGK ESRISVQERQ 206 CD2_SS1MSFPCKFVAS FLLIFNVSSK GAVSQVQLQQ SGPELEKPGA SVKLSCKASG _CD28TMYSFTGYTMNW VKQSHGKSLE WIGLITPYNG ASSYNQKFRG KATLTVDKSS _CD28_STAYMDLLSL TSEDSAVYFC ARGGYDGRGF DYWGQGTTVT VSSGGGGSGG CD40GGSGGGGSDI ELTQSPAIMS ASPGEKVTMT CSASSSVSYM HWYQQKSGTSPKRWIYDTSK LASGVPGRFS GSGSGNSYSL TISSVEAEDD ATYYCQQWSKHPLTFGAGTK LEIKAAAGSG GSGFWVLVVV GGVLACYSLL VTVAFIIFWVRSKRSRLLHS DYMNMTPRRP GPTRKHYQPY APPRDFAAYR SKKVAKKPTNKAPHPKQEPQ EINFPDDLPG SNTAAPVQET LHGCQPVTQE DGKESRISVQ ERQ 207 IL2_SS1MYRMQLLSCI ALSLALVTNS QVQLQQSGPE LEKPGASVKL SCKASGYSFT _CD28TMGYTMNWVKQS HGKSLEWIGL ITPYNGASSY NQKFRGKATL TVDKSSSTAY _CD28_MDLLSLTSED SAVYFCARGG YDGRGFDYWG QGTTVTVSSG GGGSGGGGSG CD40GGGSDIELTQ SPAIMSASPG EKVTMTCSAS SSVSYMHWYQ QKSGTSPKRWIYDTSKLASG VPGRFSGSGS GNSYSLTISS VEAEDDATYY CQQWSKHPLTFGAGTKLEIK AAAGSGGSGF WVLVVVGGVL ACYSLLVTVA FIIFWVRSKRSRLLHSDYMN MTPRRPGPTR KHYQPYAPPR DFAAYRSKKV AKKPTNKAPHPKQEPQEINF PDDLPGSNTA APVQETLHGC QPVTQEDGKE SRISVQERQ 208 GM-MWLQSLLLLG TVACSISQVQ LQQSGPELEK PGASVKLSCK ASGYSFTGYT CSF_SS1MNWVKQSHGK SLEWIGLITP YNGASSYNQK FRGKATLTVD KSSSTAYMDL _CD28TMLSLTSEDSAV YFCARGGYDG RGFDYWGQGT TVTVSSGGGG SGGGGSGGGG _CD28_SDIELTQSPA IMSASPGEKV TMTCSASSSV SYMHWYQQKS GTSPKRWIYD CD40TSKLASGVPG RFSGSGSGNS YSLTISSVEA EDDATYYCQQ WSKHPLTFGAGTKLEIKAAA GSGGSGFWVL VVVGGVLACY SLLVTVAFII FWVRSKRSRLLHSDYMNMTP RRPGPTRKHY QPYAPPRDFA AYRSKKVAKK PTNKAPHPKQEPQEINFPDD LPGSNTAAPV QETLHGCQPV TQEDGKESRI SVQERQ 209 hIgGk-MEAPAQLLFL LLLWLPDTTR QVQLQQSGPE LEKPGASVKL SCKASGYSFT VIIIGYTMNWVKQS HGKSLEWIGL ITPYNGASSY NQKFRGKATL TVDKSSSTAY _SS1_CD28MDLLSLTSED SAVYFCARGG YDGRGFDYWG QGTTVTVSSG GGGSGGGGSG TMGGGSDIELTQ SPAIMSASPG EKVTMTCSAS SSVSYMHWYQ QKSGTSPKRW _CD28_IYDTSKLASG VPGRFSGSGS GNSYSLTISS VEAEDDATYY CQQWSKHPLT CD40FGAGTKLEIK AAAGSGGSGF WVLVVVGGVL ACYSLLVTVA FIIFWVRSKRSRLLHSDYMN MTPRRPGPTR KHYQPYAPPR DFAAYRSKKV AKKPTNKAPHPKQEPQEINF PDDLPGSNTA APVQETLHGC QPVTQEDGKE SRISVQERQ 210 OSM_M5MGVLLTQRTL LSLVLALLFP SMASMQVQLV QSGAEVEKPG ASVKVSCKAS _spCD28GYTFTDYYMH WVRQAPGQGL EWMGWINPNS GGTNYAQKFQ GRVTMTRDTS _CD28_ISTAYMELSR LRSDDTAVYY CASGWDFDYW GQGTLVTVSS GGGGSGGGGS CD40GGGGSDIVMT QSPSSLSASV GDRVTITCRA SQSIRYYLSW YQQKPGKAPK CTP225LLIYTASILQ NGVPSRFSGS GSGTDFTLTI SSLQPEDFAT YYCLQTYTTPDFGPGTKVEI KAAAGSGGSG ILVKQSPMLV AYDNAVNLSC KYSYNLFSREFRASLHKGLD SAVEVCVVYG NYSQQLQVYS KTGFNCDGKL GNESVTFYLQNLYVNQTDIY FCKIEVMYPP PYLDNEKSNG TIIHVKGKHL CPSPLFPGPSKPFWVLVVVG GVLACYSLLV TVAFIIFWVR SKRSRLLHSD YMNMTPRRPGPTRKHYQPYA PPRDFAAYRS KKVAKKPTNK APHPKQEPQE INFPDDLPGSNTAAPVQETL HGCQPVTQED GKESRISVQE RQ 211 CD8a_M5MALPVTALLL PLALLLHAAR PQVQLVQSGA EVEKPGASVK VSCKASGYTF _spCD28TDYYMHWVRQ APGQGLEWMG WINPNSGGTN YAQKFQGRVT MTRDTSISTA _CD28_YMELSRLRSD DTAVYYCASG WDFDYWGQGT LVTVSSGGGG SGGGGSGGGG CD40SDIVMTQSPS SLSASVGDRV TITCRASQSI RYYLSWYQQK PGKAPKLLIYTASILQNGVP SRFSGSGSGT DFTLTISSLQ PEDFATYYCL QTYTTPDFGPGTKVEIKAAA GSGGSGILVK QSPMLVAYDN AVNLSCKYSY NLFSREFRASLHKGLDSAVE VCVVYGNYSQ QLQVYSKTGF NCDGKLGNES VTFYLQNLYVNQTDIYFCKI EVMYPPPYLD NEKSNGTIIH VKGKHLCPSP LFPGPSKPFWVLVVVGGVLA CYSLLVTVAF IIFWVRSKRS RLLHSDYMNM TPRRPGPTRKHYQPYAPPRD FAAYRSKKVA KKPTNKAPHP KQEPQEINFP DDLPGSNTAAPVQETLHGCQ PVTQEDGKES RISVQERQ 212 CD2_M5MSFPCKFVAS FLLIFNVSSK GAVSQVQLVQ SGAEVEKPGA SVKVSCKASG _spCD28YTFTDYYMHW VRQAPGQGLE WMGWINPNSG GTNYAQKFQG RVTMTRDTSI _CD28_STAYMELSRL RSDDTAVYYC ASGWDFDYWG QGTLVTVSSG GGGSGGGGSG CD40GGGSDIVMTQ SPSSLSASVG DRVTITCRAS QSIRYYLSWY QQKPGKAPKLLIYTASILQN GVPSRFSGSG SGTDFTLTIS SLQPEDFATY YCLQTYTTPDFGPGTKVEIK AAAGSGGSGI LVKQSPMLVA YDNAVNLSCK YSYNLFSREFRASLHKGLDS AVEVCVVYGN YSQQLQVYSK TGFNCDGKLG NESVTFYLQNLYVNQTDIYF CKIEVMYPPP YLDNEKSNGT IIHVKGKHLC PSPLFPGPSKPFWVLVVVGG VLACYSLLVT VAFIIFWVRS KRSRLLHSDY MNMTPRRPGPTRKHYQPYAP PRDFAAYRSK KVAKKPTNKA PHPKQEPQEI NFPDDLPGSNTAAPVQETLH GCQPVTQEDG KESRISVQER Q 213 IL2_M5MYRMQLLSCI ALSLALVTNS QVQLVQSGAE VEKPGASVKV SCKASGYTFT _spCD28DYYMHWVRQA PGQGLEWMGW INPNSGGTNY AQKFQGRVTM TRDTSISTAY _CD28_MELSRLRSDD TAVYYCASGW DFDYWGQGTL VTVSSGGGGS GGGGSGGGGS CD40DIVMTQSPSS LSASVGDRVT ITCRASQSIR YYLSWYQQKP GKAPKLLIYTASILQNGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCLQ TYTTPDFGPGTKVEIKAAAG SGGSGILVKQ SPMLVAYDNA VNLSCKYSYN LFSREFRASLHKGLDSAVEV CVVYGNYSQQ LQVYSKTGFN CDGKLGNESV TFYLQNLYVNQTDIYFCKIE VMYPPPYLDN EKSNGTIIHV KGKHLCPSPL FPGPSKPFWVLVVVGGVLAC YSLLVTVAFI IFWVRSKRSR LLHSDYMNMT PRRPGPTRKHYQPYAPPRDF AAYRSKKVAK KPTNKAPHPK QEPQEINFPD DLPGSNTAAPVQETLHGCQP VTQEDGKESR ISVQERQ 214 GM-CSF_M5MWLQSLLLLG TVACSISQVQ LVQSGAEVEK PGASVKVSCK ASGYTFTDYY _spCD28MHWVRQAPGQ GLEWMGWINP NSGGTNYAQK FQGRVTMTRD TSISTAYMEL _CD28_SRLRSDDTAV YYCASGWDFD YWGQGTLVTV SSGGGGSGGG GSGGGGSDIV CD40MTQSPSSLSA SVGDRVTITC RASQSIRYYL SWYQQKPGKA PKLLIYTASILQNGVPSRFS GSGSGTDFTL TISSLQPEDF ATYYCLQTYT TPDFGPGTKVEIKAAAGSGG SGILVKQSPM LVAYDNAVNL SCKYSYNLFS REFRASLHKGLDSAVEVCVV YGNYSQQLQV YSKTGFNCDG KLGNESVTFY LQNLYVNQTDIYFCKIEVMY PPPYLDNEKS NGTIIHVKGK HLCPSPLFPG PSKPFWVLVVVGGVLACYSL LVTVAFIIFW VRSKRSRLLH SDYMNMTPRR PGPTRKHYQPYAPPRDFAAY RSKKVAKKPT NKAPHPKQEP QEINFPDDLP GSNTAAPVQETLHGCQPVTQ EDGKESRISV QERQ 215 hIgGk-MEAPAQLLFL LLLWLPDTTR QVQLVQSGAE VEKPGASVKV SCKASGYTFT VIIIDYYMHWVRQA PGQGLEWMGW INPNSGGTNY AQKFQGRVTM TRDTSISTAY _M5MELSRLRSDD TAVYYCASGW DFDYWGQGTL VTVSSGGGGS GGGGSGGGGS _spCD28DIVMTQSPSS LSASVGDRVT ITCRASQSIR YYLSWYQQKP GKAPKLLIYT _CD28_ASILQNGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCLQ TYTTPDFGPG CD40TKVEIKAAAG SGGSGILVKQ SPMLVAYDNA VNLSCKYSYN LFSREFRASLHKGLDSAVEV CVVYGNYSQQ LQVYSKTGFN CDGKLGNESV TFYLQNLYVNQTDIYFCKIE VMYPPPYLDN EKSNGTIIHV KGKHLCPSPL FPGPSKPFWVLVVVGGVLAC YSLLVTVAFI IFWVRSKRSR LLHSDYMNMT PRRPGPTRKHYQPYAPPRDF AAYRSKKVAK KPTNKAPHPK QEPQEINFPD DLPGSNTAAPVQETLHGCQP VTQEDGKESR ISVQERQ 216 0SM_M5_spMGVLLTQRTL LSLVLALLFP SMASMQVQLV QSGAEVEKPG ASVKVSCKAS CD8GYTFTDYYMH WVRQAPGQGL EWMGWINPNS GGTNYAQKFQ GRVTMTRDTS _CD28_ISTAYMELSR LRSDDTAVYY CASGWDFDYW GQGTLVTVSS GGGGSGGGGS CD40GGGGSDIVMT QSPSSLSASV GDRVTITCRA SQSIRYYLSW YQQKPGKAPK CTP237LLIYTASILQ NGVPSRFSGS GSGTDFTLTI SSLQPEDFAT YYCLQTYTTPDFGPGTKVEI KAAAGSGGSG FVPVFLPAKP TTTPAPRPPT PAPTIASQPLSLRPEACRPA AGGAVHTRGL DFACDIYIWA PLAGTCGVLL LSLVITLYCNHRNRSKRSRL LHSDYMNMTP RRPGPTRKHY QPYAPPRDFA AYRSKKVAKKPTNKAPHPKQ EPQEINFPDD LPGSNTAAPV QETLHGCQPV TQEDGKESRI SVQERQ 217CD8a_M5_s MALPVTALLL PLALLLHAAR PQVQLVQSGA EVEKPGASVK VSCKASGYTF pCD8TDYYMHWVRQ APGQGLEWMG WINPNSGGTN YAQKFQGRVT MTRDTSISTA _CD28_YMELSRLRSD DTAVYYCASG WDFDYWGQGT LVTVSSGGGG SGGGGSGGGG CD40SDIVMTQSPS SLSASVGDRV TITCRASQSI RYYLSWYQQK PGKAPKLLIYTASILQNGVP SRFSGSGSGT DFTLTISSLQ PEDFATYYCL QTYTTPDFGPGTKVEIKAAA GSGGSGFVPV FLPAKPTTTP APRPPTPAPT IASQPLSLRPEACRPAAGGA VHTRGLDFAC DIYIWAPLAG TCGVLLLSLV ITLYCNHRNRSKRSRLLHSD YMNMTPRRPG PTRKHYQPYA PPRDFAAYRS KKVAKKPTNKAPHPKQEPQE INFPDDLPGS NTAAPVQETL HGCQPVTQED GKESRISVQE RQ 218 CD2_M5_spMSFPCKFVAS FLLIFNVSSK GAVSQVQLVQ SGAEVEKPGA SVKVSCKASG CD8YTFTDYYMHW VRQAPGQGLE WMGWINPNSG GTNYAQKFQG RVTMTRDTSI _CD28_STAYMELSRL RSDDTAVYYC ASGWDFDYWG QGTLVTVSSG GGGSGGGGSG CD40GGGSDIVMTQ SPSSLSASVG DRVTITCRAS QSIRYYLSWY QQKPGKAPKLLIYTASILQN GVPSRFSGSG SGTDFTLTIS SLQPEDFATY YCLQTYTTPDFGPGTKVEIK AAAGSGGSGF VPVFLPAKPT TTPAPRPPTP APTIASQPLSLRPEACRPAA GGAVHTRGLD FACDIYIWAP LAGTCGVLLL SLVITLYCNHRNRSKRSRLL HSDYMNMTPR RPGPTRKHYQ PYAPPRDFAA YRSKKVAKKPTNKAPHPKQE PQEINFPDDL PGSNTAAPVQ ETLHGCQPVT QEDGKESRIS VQERQ 219IL2_M5_sp MYRMQLLSCI ALSLALVTNS QVQLVQSGAE VEKPGASVKV SCKASGYTFT CD8DYYMHWVRQA PGQGLEWMGW INPNSGGTNY AQKFQGRVTM TRDTSISTAY _CD28_MELSRLRSDD TAVYYCASGW DFDYWGQGTL VTVSSGGGGS GGGGSGGGGS CD40DIVMTQSPSS LSASVGDRVT ITCRASQSIR YYLSWYQQKP GKAPKLLIYTASILQNGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCLQ TYTTPDFGPGTKVEIKAAAG SGGSGFVPVF LPAKPTTTPA PRPPTPAPTI ASQPLSLRPEACRPAAGGAV HTRGLDFACD IYIWAPLAGT CGVLLLSLVI TLYCNHRNRSKRSRLLHSDY MNMTPRRPGP TRKHYQPYAP PRDFAAYRSK KVAKKPTNKAPHPKQEPQEI NFPDDLPGSN TAAPVQETLH GCQPVTQEDG KESRISVQER Q 220 GM-MWLQSLLLLG TVACSISQVQ LVQSGAEVEK PGASVKVSCK ASGYTFTDYY CSF_M5_spMHWVRQAPGQ GLEWMGWINP NSGGTNYAQK FQGRVTMTRD TSISTAYMEL CD8SRLRSDDTAV YYCASGWDFD YWGQGTLVTV SSGGGGSGGG GSGGGGSDIV _CD28_MTQSPSSLSA SVGDRVTITC RASQSIRYYL SWYQQKPGKA PKLLIYTASI CD40LQNGVPSRFS GSGSGTDFTL TISSLQPEDF ATYYCLQTYT TPDFGPGTKVEIKAAAGSGG SGFVPVFLPA KPTTTPAPRP PTPAPTIASQ PLSLRPEACRPAAGGAVHTR GLDFACDIYI WAPLAGTCGV LLLSLVITLY CNHRNRSKRSRLLHSDYMNM TPRRPGPTRK HYQPYAPPRD FAAYRSKKVA KKPTNKAPHPKQEPQEINFP DDLPGSNTAA PVQETLHGCQ PVTQEDGKES RISVQERQ 221 hIgGk-MEAPAQLLFL LLLWLPDTTR QVQLVQSGAE VEKPGASVKV SCKASGYTFT VIIIDYYMHWVRQA PGQGLEWMGW INPNSGGTNY AQKFQGRVTM TRDTSISTAY _M5_spCD8MELSRLRSDD TAVYYCASGW DFDYWGQGTL VTVSSGGGGS GGGGSGGGGS _CD28_DIVMTQSPSS LSASVGDRVT ITCRASQSIR YYLSWYQQKP GKAPKLLIYT CD40ASILQNGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCLQ TYTTPDFGPGTKVEIKAAAG SGGSGFVPVF LPAKPTTTPA PRPPTPAPTI ASQPLSLRPEACRPAAGGAV HTRGLDFACD IYIWAPLAGT CGVLLLSLVI TLYCNHRNRSKRSRLLHSDY MNMTPRRPGP TRKHYQPYAP PRDFAAYRSK KVAKKPTNKAPHPKQEPQEI NFPDDLPGSN TAAPVQETLH GCQPVTQEDG KESRISVQER Q 222 OSM_M5_CDMGVLLTQRTL LSLVLALLFP SMASMQVQLV QSGAEVEKPG ASVKVSCKAS 28TMGYTFTDYYMH WVRQAPGQGL EWMGWINPNS GGTNYAQKFQ GRVTMTRDTS _CD28_ISTAYMELSR LRSDDTAVYY CASGWDFDYW GQGTLVTVSS GGGGSGGGGS CD40GGGGSDIVMT QSPSSLSASV GDRVTITCRA SQSIRYYLSW YQQKPGKAPKLLIYTASILQ NGVPSRFSGS GSGTDFTLTI SSLQPEDFAT YYCLQTYTTPDFGPGTKVEI KAAAGSGGSG FWVLVVVGGV LACYSLLVTV AFIIFWVRSKRSRLLHSDYM NMTPRRPGPT RKHYQPYAPP RDFAAYRSKK VAKKPTNKAPHPKQEPQEIN FPDDLPGSNT AAPVQETLHG CQPVTQEDGK ESRISVQERQ 223 CD8a_M5_CMALPVTALLL PLALLLHAAR PQVQLVQSGA EVEKPGASVK VSCKASGYTF D28TMTDYYMHWVRQ APGQGLEWMG WINPNSGGTN YAQKFQGRVT MTRDTSISTA _CD28_YMELSRLRSD DTAVYYCASG WDFDYWGQGT LVTVSSGGGG SGGGGSGGGG CD40SDIVMTQSPS SLSASVGDRV TITCRASQSI RYYLSWYQQK PGKAPKLLIYTASILQNGVP SRFSGSGSGT DFTLTISSLQ PEDFATYYCL QTYTTPDFGPGTKVEIKAAA GSGGSGFWVL VVVGGVLACY SLLVTVAFII FWVRSKRSRLLHSDYMNMTP RRPGPTRKHY QPYAPPRDFA AYRSKKVAKK PTNKAPHPKQEPQEINFPDD LPGSNTAAPV QETLHGCQPV TQEDGKESRI SVQERQ 224 CD2_M5_CDMSFPCKFVAS FLLIFNVSSK GAVSQVQLVQ SGAEVEKPGA SVKVSCKASG 28TMYTFTDYYMHW VRQAPGQGLE WMGWINPNSG GTNYAQKFQG RVTMTRDTSI _CD28_STAYMELSRL RSDDTAVYYC ASGWDFDYWG QGTLVTVSSG GGGSGGGGSG CD40GGGSDIVMTQ SPSSLSASVG DRVTITCRAS QSIRYYLSWY QQKPGKAPKLLIYTASILQN GVPSRFSGSG SGTDFTLTIS SLQPEDFATY YCLQTYTTPDFGPGTKVEIK AAAGSGGSGF WVLVVVGGVL ACYSLLVTVA FIIFWVRSKRSRLLHSDYMN MTPRRPGPTR KHYQPYAPPR DFAAYRSKKV AKKPTNKAPHPKQEPQEINF PDDLPGSNTA APVQETLHGC QPVTQEDGKE SRISVQERQ 225 IL2_M5_CDMYRMQLLSCI ALSLALVTNS QVQLVQSGAE VEKPGASVKV SCKASGYTFT 28TMDYYMHWVRQA PGQGLEWMGW INPNSGGTNY AQKFQGRVTM TRDTSISTAY _CD28_MELSRLRSDD TAVYYCASGW DFDYWGQGTL VTVSSGGGGS GGGGSGGGGS CD40DIVMTQSPSS LSASVGDRVT ITCRASQSIR YYLSWYQQKP GKAPKLLIYTASILQNGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCLQ TYTTPDFGPGTKVEIKAAAG SGGSGFWVLV VVGGVLACYS LLVTVAFIIF WVRSKRSRLLHSDYMNMTPR RPGPTRKHYQ PYAPPRDFAA YRSKKVAKKP TNKAPHPKQEPQEINFPDDL PGSNTAAPVQ ETLHGCQPVT QEDGKESRIS VQERQ 226 GM-CSF_M5MWLQSLLLLG TVACSISQVQ LVQSGAEVEK PGASVKVSCK ASGYTFTDYY _CD28TMMHWVRQAPGQ GLEWMGWINP NSGGTNYAQK FQGRVTMTRD TSISTAYMEL _CD28_SRLRSDDTAV YYCASGWDFD YWGQGTLVTV SSGGGGSGGG GSGGGGSDIV CD40MTQSPSSLSA SVGDRVTITC RASQSIRYYL SWYQQKPGKA PKLLIYTASILQNGVPSRFS GSGSGTDFTL TISSLQPEDF ATYYCLQTYT TPDFGPGTKVEIKAAAGSGG SGFWVLVVVG GVLACYSLLV TVAFIIFWVR SKRSRLLHSDYMNMTPRRPG PTRKHYQPYA PPRDFAAYRS KKVAKKPTNK APHPKQEPQEINFPDDLPGS NTAAPVQETL HGCQPVTQED GKESRISVQE RQ 227 hIgGk-MEAPAQLLFL LLLWLPDTTR QVQLVQSGAE VEKPGASVKV SCKASGYTFT VIIIDYYMHWVRQA PGQGLEWMGW INPNSGGTNY AQKFQGRVTM TRDTSISTAY _M5_MELSRLRSDD TAVYYCASGW DFDYWGQGTL VTVSSGGGGS GGGGSGGGGS CD28TMDIVMTQSPSS LSASVGDRVT ITCRASQSIR YYLSWYQQKP GKAPKLLIYT _CD28_ASILQNGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCLQ TYTTPDFGPG CD40TKVEIKAAAG SGGSGFWVLV VVGGVLACYS LLVTVAFIIF WVRSKRSRLLHSDYMNMTPR RPGPTRKHYQ PYAPPRDFAA YRSKKVAKKP TNKAPHPKQEPQEINFPDDL PGSNTAAPVQ ETLHGCQPVT QEDGKESRIS VQERQ 228 OSM_HN1MGVLLTQRTL LSLVLALLFP SMASMQVQLV QSGAEVKRPG ASVQVSCRAS _spCD28GYSINTYYMQ WVRQAPGAGL EWMGVINPSG VTSYAQKFQG RVTLTNDTST _CD28_NTVYMQLNSL TSADTAVYYC ARWALWGDFG MDVWGKGTLV TVSSGGGGSG CD40GGGSGGGGSD IQMTQSPSTL SASIGDRVTI TCRASEGIYH WLAWYQQKPG CTP226KAPKLLIYKA SSLASGAPSR FSGSGSGTDF TLTISSLQPD DFATYYCQQYSNYPLTFGGG TKLEIKAAAG SGGSGILVKQ SPMLVAYDNA VNLSCKYSYNLFSREFRASL HKGLDSAVEV CVVYGNYSQQ LQVYSKTGFN CDGKLGNESVTFYLQNLYVN QTDIYFCKIE VMYPPPYLDN EKSNGTIIHV KGKHLCPSPLFPGPSKPFWV LVVVGGVLAC YSLLVTVAFI IFWVRSKRSR LLHSDYMNMTPRRPGPTRKH YQPYAPPRDF AAYRSKKVAK KPTNKAPHPK QEPQEINFPDDLPGSNTAAP VQETLHGCQP VTQEDGKESR ISVQERQ 229 CD8a_HNlMALPVTALLL PLALLLHAAR PQVQLVQSGA EVKRPGASVQ VSCRASGYSI _spCD28NTYYMQWVRQ APGAGLEWMG VINPSGVTSY AQKFQGRVTL TNDTSTNTVY _CD28_MQLNSLTSAD TAVYYCARWA LWGDFGMDVW GKGTLVTVSS GGGGSGGGGS CD40GGGGSDIQMT QSPSTLSASI GDRVTITCRA SEGIYHWLAW YQQKPGKAPKLLIYKASSLA SGAPSRFSGS GSGTDFTLTI SSLQPDDFAT YYCQQYSNYPLTFGGGTKLE IKAAAGSGGS GILVKQSPML VAYDNAVNLS CKYSYNLFSREFRASLHKGL DSAVEVCVVY GNYSQQLQVY SKTGFNCDGK LGNESVTFYLQNLYVNQTDI YFCKIEVMYP PPYLDNEKSN GTIIHVKGKH LCPSPLFPGPSKPFWVLVVV GGVLACYSLL VTVAFIIFWV RSKRSRLLHS DYMNMTPRRPGPTRKHYQPY APPRDFAAYR SKKVAKKPTN KAPHPKQEPQ EINFPDDLPGSNTAAPVQET LHGCQPVTQE DGKESRISVQ ERQ 230 CD2_HN1MSFPCKFVAS FLLIFNVSSK GAVSQVQLVQ SGAEVKRPGA SVQVSCRASG _spCD28YSINTYYMQW VRQAPGAGLE WMGVINPSGV TSYAQKFQGR VTLTNDTSTN _CD28_TVYMQLNSLT SADTAVYYCA RWALWGDFGM DVWGKGTLVT VSSGGGGSGG CD40GGSGGGGSDI QMTQSPSTLS ASIGDRVTIT CRASEGIYHW LAWYQQKPGKAPKLLIYKAS SLASGAPSRF SGSGSGTDFT LTISSLQPDD FATYYCQQYSNYPLTFGGGT KLEIKAAAGS GGSGILVKQS PMLVAYDNAV NLSCKYSYNLFSREFRASLH KGLDSAVEVC VVYGNYSQQL QVYSKTGFNC DGKLGNESVTFYLQNLYVNQ TDIYFCKIEV MYPPPYLDNE KSNGTIIHVK GKHLCPSPLFPGPSKPFWVL VVVGGVLACY SLLVTVAFII FWVRSKRSRL LHSDYMNMTPRRPGPTRKHY QPYAPPRDFA AYRSKKVAKK PTNKAPHPKQ EPQEINFPDDLPGSNTAAPV QETLHGCQPV TQEDGKESRI SVQERQ 231 IL2_HN1MYRMQLLSCI ALSLALVTNS QVQLVQSGAE VKRPGASVQV SCRASGYSIN _spCD28TYYMQWVRQA PGAGLEWMGV INPSGVTSYA QKFQGRVTLT NDTSTNTVYM _CD28_QLNSLTSADT AVYYCARWAL WGDFGMDVWG KGTLVTVSSG GGGSGGGGSG CD40GGGSDIQMTQ SPSTLSASIG DRVTITCRAS EGIYHWLAWY QQKPGKAPKLLIYKASSLAS GAPSRFSGSG SGTDFTLTIS SLQPDDFATY YCQQYSNYPLTFGGGTKLEI KAAAGSGGSG ILVKQSPMLV AYDNAVNLSC KYSYNLFSREFRASLHKGLD SAVEVCVVYG NYSQQLQVYS KTGFNCDGKL GNESVTFYLQNLYVNQTDIY FCKIEVMYPP PYLDNEKSNG TIIHVKGKHL CPSPLFPGPSKPFWVLVVVG GVLACYSLLV TVAFIIFWVR SKRSRLLHSD YMNMTPRRPGPTRKHYQPYA PPRDFAAYRS KKVAKKPTNK APHPKQEPQE INFPDDLPGSNTAAPVQETL HGCQPVTQED GKESRISVQE RQ 232 GM-MWLQSLLLLG TVACSISQVQ LVQSGAEVKR PGASVQVSCR ASGYSINTYY CSF_HN1MQWVRQAPGA GLEWMGVINP SGVTSYAQKF QGRVTLTNDT STNTVYMQLN _spCD28SLTSADTAVY YCARWALWGD FGMDVWGKGT LVTVSSGGGG SGGGGSGGGG _CD28_SDIQMTQSPS TLSASIGDRV TITCRASEGI YHWLAWYQQK PGKAPKLLIY CD40KASSLASGAP SRFSGSGSGT DFTLTISSLQ PDDFATYYCQ QYSNYPLTFGGGTKLEIKAA AGSGGSGILV KQSPMLVAYD NAVNLSCKYS YNLFSREFRASLHKGLDSAV EVCVVYGNYS QQLQVYSKTG FNCDGKLGNE SVTFYLQNLYVNQTDIYFCK IEVMYPPPYL DNEKSNGTII HVKGKHLCPS PLFPGPSKPFWVLVVVGGVL ACYSLLVTVA FIIFWVRSKR SRLLHSDYMN MTPRRPGPTRKHYQPYAPPR DFAAYRSKKV AKKPTNKAPH PKQEPQEINF PDDLPGSNTAAPVQETLHGC QPVTQEDGKE SRISVQERQ 233 hIgGk-MEAPAQLLFL LLLWLPDTTR QVQLVQSGAE VKRPGASVQV SCRASGYSIN VIIITYYMQWVRQA PGAGLEWMGV INPSGVTSYA QKFQGRVTLT NDTSTNTVYM _HN1QLNSLTSADT AVYYCARWAL WGDFGMDVWG KGTLVTVSSG GGGSGGGGSG _spCD28GGGSDIQMTQ SPSTLSASIG DRVTITCRAS EGIYHWLAWY QQKPGKAPKL _CD28_LIYKASSLAS GAPSRFSGSG SGTDFTLTIS SLQPDDFATY YCQQYSNYPL CD40TFGGGTKLEI KAAAGSGGSG ILVKQSPMLV AYDNAVNLSC KYSYNLFSREFRASLHKGLD SAVEVCVVYG NYSQQLQVYS KTGFNCDGKL GNESVTFYLQNLYVNQTDIY FCKIEVMYPP PYLDNEKSNG TIIHVKGKHL CPSPLFPGPSKPFWVLVVVG GVLACYSLLV TVAFIIFWVR SKRSRLLHSD YMNMTPRRPGPTRKHYQPYA PPRDFAAYRS KKVAKKPTNK APHPKQEPQE INFPDDLPGSNTAAPVQETL HGCQPVTQED GKESRISVQE RQ 234 OSM_HN1_sMGVLLTQRTL LSLVLALLFP SMASMQVQLV QSGAEVKRPG ASVQVSCRAS pCD8GYSINTYYMQ WVRQAPGAGL EWMGVINPSG VTSYAQKFQG RVTLTNDTST _CD28_NTVYMQLNSL TSADTAVYYC ARWALWGDFG MDVWGKGTLV TVSSGGGGSG CD40GGGSGGGGSD IQMTQSPSTL SASIGDRVTI TCRASEGIYH WLAWYQQKPG CTP238KAPKLLIYKA SSLASGAPSR FSGSGSGTDF TLTISSLQPD DFATYYCQQYSNYPLTFGGG TKLEIKAAAG SGGSGFVPVF LPAKPTTTPA PRPPTPAPTIASQPLSLRPE ACRPAAGGAV HTRGLDFACD IYIWAPLAGT CGVLLLSLVITLYCNHRNRS KRSRLLHSDY MNMTPRRPGP TRKHYQPYAP PRDFAAYRSKKVAKKPTNKA PHPKQEPQEI NFPDDLPGSN TAAPVQETLH GCQPVTQEDG KESRISVQER Q 235CD8a_HN1_ MALPVTALLL PLALLLHAAR PQVQLVQSGA EVKRPGASVQ VSCRASGYSI spCD8NTYYMQWVRQ APGAGLEWMG VINPSGVTSY AQKFQGRVTL TNDTSTNTVY _CD28_MQLNSLTSAD TAVYYCARWA LWGDFGMDVW GKGTLVTVSS GGGGSGGGGS CD40GGGGSDIQMT QSPSTLSASI GDRVTITCRA SEGIYHWLAW YQQKPGKAPKLLIYKASSLA SGAPSRFSGS GSGTDFTLTI SSLQPDDFAT YYCQQYSNYPLTFGGGTKLE IKAAAGSGGS GFVPVFLPAK PTTTPAPRPP TPAPTIASQPLSLRPEACRP AAGGAVHTRG LDFACDIYIW APLAGTCGVL LLSLVITLYCNHRNRSKRSR LLHSDYMNMT PRRPGPTRKH YQPYAPPRDF AAYRSKKVAKKPTNKAPHPK QEPQEINFPD DLPGSNTAAP VQETLHGCQP VTQEDGKESR ISVQERQ 236CD2_HN1_s MSFPCKFVAS FLLIFNVSSK GAVSQVQLVQ SGAEVKRPGA SVQVSCRASG pCD8YSINTYYMQW VRQAPGAGLE WMGVINPSGV TSYAQKFQGR VTLTNDTSTN _CD28_TVYMQLNSLT SADTAVYYCA RWALWGDFGM DVWGKGTLVT VSSGGGGSGG CD40GGSGGGGSDI QMTQSPSTLS ASIGDRVTIT CRASEGIYHW LAWYQQKPGKAPKLLIYKAS SLASGAPSRF SGSGSGTDFT LTISSLQPDD FATYYCQQYSNYPLTFGGGT KLEIKAAAGS GGSGFVPVFL PAKPTTTPAP RPPTPAPTIASQPLSLRPEA CRPAAGGAVH TRGLDFACDI YIWAPLAGTC GVLLLSLVITLYCNHRNRSK RSRLLHSDYM NMTPRRPGPT RKHYQPYAPP RDFAAYRSKKVAKKPTNKAP HPKQEPQEIN FPDDLPGSNT AAPVQETLHG CQPVTQEDGK ESRISVQERQ 237IL2_HN1_s MYRMQLLSCI ALSLALVTNS QVQLVQSGAE VKRPGASVQV SCRASGYSIN pCD8TYYMQWVRQA PGAGLEWMGV INPSGVTSYA QKFQGRVTLT NDTSTNTVYM _CD28_QLNSLTSADT AVYYCARWAL WGDFGMDVWG KGTLVTVSSG GGGSGGGGSG CD40GGGSDIQMTQ SPSTLSASIG DRVTITCRAS EGIYHWLAWY QQKPGKAPKLLIYKASSLAS GAPSRFSGSG SGTDFTLTIS SLQPDDFATY YCQQYSNYPLTFGGGTKLEI KAAAGSGGSG FVPVFLPAKP TTTPAPRPPT PAPTIASQPLSLRPEACRPA AGGAVHTRGL DFACDIYIWA PLAGTCGVLL LSLVITLYCNHRNRSKRSRL LHSDYMNMTP RRPGPTRKHY QPYAPPRDFA AYRSKKVAKKPTNKAPHPKQ EPQEINFPDD LPGSNTAAPV QETLHGCQPV TQEDGKESRI SVQERQ 238 GM-MWLQSLLLLG TVACSISQVQ LVQSGAEVKR PGASVQVSCR ASGYSINTYY CSF_HN1MQWVRQAPGA GLEWMGVINP SGVTSYAQKF QGRVTLTNDT STNTVYMQLN _spCD8SLTSADTAVY YCARWALWGD FGMDVWGKGT LVTVSSGGGG SGGGGSGGGG _CD28_SDIQMTQSPS TLSASIGDRV TITCRASEGI YHWLAWYQQK PGKAPKLLIY CD40KASSLASGAP SRFSGSGSGT DFTLTISSLQ PDDFATYYCQ QYSNYPLTFGGGTKLEIKAA AGSGGSGFVP VFLPAKPTTT PAPRPPTPAP TIASQPLSLRPEACRPAAGG AVHTRGLDFA CDIYIWAPLA GTCGVLLLSL VITLYCNHRNRSKRSRLLHS DYMNMTPRRP GPTRKHYQPY APPRDFAAYR SKKVAKKPTNKAPHPKQEPQ EINFPDDLPG SNTAAPVQET LHGCQPVTQE DGKESRISVQ ERQ 239 hIgGk-MEAPAQLLFL LLLWLPDTTR QVQLVQSGAE VKRPGASVQV SCRASGYSIN VIIITYYMQWVRQA PGAGLEWMGV INPSGVTSYA QKFQGRVTLT NDTSTNTVYM _HN1_spCDQLNSLTSADT AVYYCARWAL WGDFGMDVWG KGTLVTVSSG GGGSGGGGSG 8GGGSDIQMTQ SPSTLSASIG DRVTITCRAS EGIYHWLAWY QQKPGKAPKL _CD28_LIYKASSLAS GAPSRFSGSG SGTDFTLTIS SLQPDDFATY YCQQYSNYPL CD40TFGGGTKLEI KAAAGSGGSG FVPVFLPAKP TTTPAPRPPT PAPTIASQPLSLRPEACRPA AGGAVHTRGL DFACDIYIWA PLAGTCGVLL LSLVITLYCNHRNRSKRSRL LHSDYMNMTP RRPGPTRKHY QPYAPPRDFA AYRSKKVAKKPTNKAPHPKQ EPQEINFPDD LPGSNTAAPV QETLHGCQPV TQEDGKESRI SVQERQ 240OSM_HN1_C MGVLLTQRTL LSLVLALLFP SMASMQVQLV QSGAEVKRPG ASVQVSCRAS D28TMGYSINTYYMQ WVRQAPGAGL EWMGVINPSG VTSYAQKFQG RVTLTNDTST _CD28_NTVYMQLNSL TSADTAVYYC ARWALWGDFG MDVWGKGTLV TVSSGGGGSG CD40GGGSGGGGSD IQMTQSPSTL SASIGDRVTI TCRASEGIYH WLAWYQQKPGKAPKLLIYKA SSLASGAPSR FSGSGSGTDF TLTISSLQPD DFATYYCQQYSNYPLTFGGG TKLEIKAAAG SGGSGFWVLV VVGGVLACYS LLVTVAFIIFWVRSKRSRLL HSDYMNMTPR RPGPTRKHYQ PYAPPRDFAA YRSKKVAKKPTNKAPHPKQE PQEINFPDDL PGSNTAAPVQ ETLHGCQPVT QEDGKESRISVQERQRSKRS RLLHSDYMNM TPRRPGPTRK HYQPYAPPRD FAAYRSKKVAKKPTNKAPHP KQEPQEINFP DDLPGSNTAA PVQETLHGCQ PVTQEDGKES RISVQERQ 241CD8a_HN1_ MALPVTALLL PLALLLHAAR PQVQLVQSGA EVKRPGASVQ VSCRASGYSI CD28TMNTYYMQWVRQ APGAGLEWMG VINPSGVTSY AQKFQGRVTL TNDTSTNTVY _CD28_MQLNSLTSAD TAVYYCARWA LWGDFGMDVW GKGTLVTVSS GGGGSGGGGS CD40GGGGSDIQMT QSPSTLSASI GDRVTITCRA SEGIYHWLAW YQQKPGKAPKLLIYKASSLA SGAPSRFSGS GSGTDFTLTI SSLQPDDFAT YYCQQYSNYPLTFGGGTKLE IKAAAGSGGS GFWVLVVVGG VLACYSLLVT VAFIIFWVRSKRSRLLHSDY MNMTPRRPGP TRKHYQPYAP PRDFAAYRSK KVAKKPTNKAPHPKQEPQEI NFPDDLPGSN TAAPVQETLH GCQPVTQEDG KESRISVQER Q 242 CD2_HN1_CMSFPCKFVAS FLLIFNVSSK GAVSQVQLVQ SGAEVKRPGA SVQVSCRASG D28TMYSINTYYMQW VRQAPGAGLE WMGVINPSGV TSYAQKFQGR VTLTNDTSTN _CD28_TVYMQLNSLT SADTAVYYCA RWALWGDFGM DVWGKGTLVT VSSGGGGSGG CD40GGSGGGGSDI QMTQSPSTLS ASIGDRVTIT CRASEGIYHW LAWYQQKPGKAPKLLIYKAS SLASGAPSRF SGSGSGTDFT LTISSLQPDD FATYYCQQYSNYPLTFGGGT KLEIKAAAGS GGSGFWVLVV VGGVLACYSL LVTVAFIIFWVRSKRSRLLH SDYMNMTPRR PGPTRKHYQP YAPPRDFAAY RSKKVAKKPTNKAPHPKQEP QEINFPDDLP GSNTAAPVQE TLHGCQPVTQ EDGKESRISV QERQ 243IL2_HN1_C MYRMQLLSCI ALSLALVTNS QVQLVQSGAE VKRPGASVQV SCRASGYSIN D28TMTYYMQWVRQA PGAGLEWMGV INPSGVTSYA QKFQGRVTLT NDTSTNTVYM _CD28_QLNSLTSADT AVYYCARWAL WGDFGMDVWG KGTLVTVSSG GGGSGGGGSG CD40GGGSDIQMTQ SPSTLSASIG DRVTITCRAS EGIYHWLAWY QQKPGKAPKLLIYKASSLAS GAPSRFSGSG SGTDFTLTIS SLQPDDFATY YCQQYSNYPLTFGGGTKLEI KAAAGSGGSG FWVLVVVGGV LACYSLLVTV AFIIFWVRSKRSRLLHSDYM NMTPRRPGPT RKHYQPYAPP RDFAAYRSKK VAKKPTNKAPHPKQEPQEIN FPDDLPGSNT AAPVQETLHG CQPVTQEDGK ESRISVQERQ 244 GM-MWLQSLLLLG TVACSISQVQ LVQSGAEVKR PGASVQVSCR ASGYSINTYY CSF_HN1MQWVRQAPGA GLEWMGVINP SGVTSYAQKF QGRVTLTNDT STNTVYMQLN _CD28TMSLTSADTAVY YCARWALWGD FGMDVWGKGT LVTVSSGGGG SGGGGSGGGG _CD28_SDIQMTQSPS TLSASIGDRV TITCRASEGI YHWLAWYQQK PGKAPKLLIY CD40KASSLASGAP SRFSGSGSGT DFTLTISSLQ PDDFATYYCQ QYSNYPLTFGGGTKLEIKAA AGSGGSGFWV LVVVGGVLAC YSLLVTVAFI IFWVRSKRSRLLHSDYMNMT PRRPGPTRKH YQPYAPPRDF AAYRSKKVAK KPTNKAPHPKQEPQEINFPD DLPGSNTAAP VQETLHGCQP VTQEDGKESR ISVQERQ 245 hIgGk-MEAPAQLLFL LLLWLPDTTR QVQLVQSGAE VKRPGASVQV SCRASGYSIN VIIITYYMQWVRQA PGAGLEWMGV INPSGVTSYA QKFQGRVTLT NDTSTNTVYM _HN1_CD28QLNSLTSADT AVYYCARWAL WGDFGMDVWG KGTLVTVSSG GGGSGGGGSG TMGGGSDIQMTQ SPSTLSASIG DRVTITCRAS EGIYHWLAWY QQKPGKAPKL _CD28_LIYKASSLAS GAPSRFSGSG SGTDFTLTIS SLQPDDFATY YCQQYSNYPL CD40TFGGGTKLEI KAAAGSGGSG FWVLVVVGGV LACYSLLVTV AFIIFWVRSKRSRLLHSDYM NMTPRRPGPT RKHYQPYAPP RDFAAYRSKK VAKKPTNKAPHPKQEPQEIN FPDDLPGSNT AAPVQETLHG CQPVTQEDGK ESRISVQERQ 246 OSM_M912MGVLLTQRTL LSLVLALLFP SMASMQVQLQ ESGPGLVKPS ETLSLTCTVS _spCD28GGSVSSGSYY WSWIRQPPGK GLEWIGYIYY SGSTNYNPSL KSRVTISVDT _CD28_SKNQFSLKLS SVTAADTAVY YCAREGKNGA FDIWGQGTMV TVSSGGGGSG CD40GGGSGGGGSD IQMTQSPSSL SASVGDRVTI TCRASQSISS YLNWYQQKPG CTP227KAPKLLIYAA SSLQSGVPSG FSGSGSGTDF TLTISSLQPE DFATYYCQQSYSTPLTFGGG TKVEIKAAAG SGGSGILVKQ SPMLVAYDNA VNLSCKYSYNLFSREFRASL HKGLDSAVEV CVVYGNYSQQ LQVYSKTGFN CDGKLGNESVTFYLQNLYVN QTDIYFCKIE VMYPPPYLDN EKSNGTIIHV KGKHLCPSPLFPGPSKPFWV LVVVGGVLAC YSLLVTVAFI IFWVRSKRSR LLHSDYMNMTPRRPGPTRKH YQPYAPPRDF AAYRSKKVAK KPTNKAPHPK QEPQEINFPDDLPGSNTAAP VQETLHGCQP VTQEDGKESR ISVQERQ 247 CD8a_M912MALPVTALLL PLALLLHAAR PQVQLQESGP GLVKPSETLS LTCTVSGGSV _spCD28SSGSYYWSWI RQPPGKGLEW IGYIYYSGST NYNPSLKSRV TISVDTSKNQ _CD28_FSLKLSSVTA ADTAVYYCAR EGKNGAFDIW GQGTMVTVSS GGGGSGGGGS CD40GGGGSDIQMT QSPSSLSASV GDRVTITCRA SQSISSYLNW YQQKPGKAPKLLIYAASSLQ SGVPSGFSGS GSGTDFTLTI SSLQPEDFAT YYCQQSYSTPLTFGGGTKVE IKAAAGSGGS GILVKQSPML VAYDNAVNLS CKYSYNLFSREFRASLHKGL DSAVEVCVVY GNYSQQLQVY SKTGFNCDGK LGNESVTFYLQNLYVNQTDI YFCKIEVMYP PPYLDNEKSN GTIIHVKGKH LCPSPLFPGPSKPFWVLVVV GGVLACYSLL VTVAFIIFWV RSKRSRLLHS DYMNMTPRRPGPTRKHYQPY APPRDFAAYR SKKVAKKPTN KAPHPKQEPQ EINFPDDLPGSNTAAPVQET LHGCQPVTQE DGKESRISVQ ERQ 248 CD2_M912MSFPCKFVAS FLLIFNVSSK GAVSQVQLQE SGPGLVKPSE TLSLTCTVSG _spCD28GSVSSGSYYW SWIRQPPGKG LEWIGYIYYS GSTNYNPSLK SRVTISVDTS _CD28_KNQFSLKLSS VTAADTAVYY CAREGKNGAF DIWGQGTMVT VSSGGGGSGG CD40GGSGGGGSDI QMTQSPSSLS ASVGDRVTIT CRASQSISSY LNWYQQKPGKAPKLLIYAAS SLQSGVPSGF SGSGSGTDFT LTISSLQPED FATYYCQQSYSTPLTFGGGT KVEIKAAAGS GGSGILVKQS PMLVAYDNAV NLSCKYSYNLFSREFRASLH KGLDSAVEVC VVYGNYSQQL QVYSKTGFNC DGKLGNESVTFYLQNLYVNQ TDIYFCKIEV MYPPPYLDNE KSNGTIIHVK GKHLCPSPLFPGPSKPFWVL VVVGGVLACY SLLVTVAFII FWVRSKRSRL LHSDYMNMTPRRPGPTRKHY QPYAPPRDFA AYRSKKVAKK PTNKAPHPKQ EPQEINFPDDLPGSNTAAPV QETLHGCQPV TQEDGKESRI SVQERQ 249 IL2_M912MYRMQLLSCI ALSLALVTNS QVQLQESGPG LVKPSETLSL TCTVSGGSVS _spCD28SGSYYWSWIR QPPGKGLEWI GYIYYSGSTN YNPSLKSRVT ISVDTSKNQF _CD28_SLKLSSVTAA DTAVYYCARE GKNGAFDIWG QGTMVTVSSG GGGSGGGGSG CD40GGGSDIQMTQ SPSSLSASVG DRVTITCRAS QSISSYLNWY QQKPGKAPKLLIYAASSLQS GVPSGFSGSG SGTDFTLTIS SLQPEDFATY YCQQSYSTPLTFGGGTKVEI KAAAGSGGSG ILVKQSPMLV AYDNAVNLSC KYSYNLFSREFRASLHKGLD SAVEVCVVYG NYSQQLQVYS KTGFNCDGKL GNESVTFYLQNLYVNQTDIY FCKIEVMYPP PYLDNEKSNG TIIHVKGKHL CPSPLFPGPSKPFWVLVVVG GVLACYSLLV TVAFIIFWVR SKRSRLLHSD YMNMTPRRPGPTRKHYQPYA PPRDFAAYRS KKVAKKPTNK APHPKQEPQE INFPDDLPGSNTAAPVQETL HGCQPVTQED GKESRISVQE RQ 250 GM-MWLQSLLLLG TVACSISQVQ LQESGPGLVK PSETLSLTCT VSGGSVSSGS CSF_M912YYWSWIRQPP GKGLEWIGYI YYSGSTNYNP SLKSRVTISV DTSKNQFSLK _spCD28LSSVTAADTA VYYCAREGKN GAFDIWGQGT MVTVSSGGGG SGGGGSGGGG _CD28_SDIQMTQSPS SLSASVGDRV TITCRASQSI SSYLNWYQQK PGKAPKLLIY CD40AASSLQSGVP SGFSGSGSGT DFTLTISSLQ PEDFATYYCQ QSYSTPLTFGGGTKVEIKAA AGSGGSGILV KQSPMLVAYD NAVNLSCKYS YNLFSREFRASLHKGLDSAV EVCVVYGNYS QQLQVYSKTG FNCDGKLGNE SVTFYLQNLYVNQTDIYFCK IEVMYPPPYL DNEKSNGTII HVKGKHLCPS PLFPGPSKPFWVLVVVGGVL ACYSLLVTVA FIIFWVRSKR SRLLHSDYMN MTPRRPGPTRKHYQPYAPPR DFAAYRSKKV AKKPTNKAPH PKQEPQEINF PDDLPGSNTAAPVQETLHGC QPVTQEDGKE SRISVQERQ 251 hIgGk-MEAPAQLLFL LLLWLPDTTR QVQLQESGPG LVKPSETLSL TCTVSGGSVS VIIISGSYYWSWIR QPPGKGLEWI GYIYYSGSTN YNPSLKSRVT ISVDTSKNQF _M912SLKLSSVTAA DTAVYYCARE GKNGAFDIWG QGTMVTVSSG GGGSGGGGSG _spCD28GGGSDIQMTQ SPSSLSASVG DRVTITCRAS QSISSYLNWY QQKPGKAPKL _CD28_LIYAASSLQS GVPSGFSGSG SGTDFTLTIS SLQPEDFATY YCQQSYSTPL CD40TFGGGTKVEI KAAAGSGGSG ILVKQSPMLV AYDNAVNLSC KYSYNLFSREFRASLHKGLD SAVEVCVVYG NYSQQLQVYS KTGFNCDGKL GNESVTFYLQNLYVNQTDIY FCKIEVMYPP PYLDNEKSNG TIIHVKGKHL CPSPLFPGPSKPFWVLVVVG GVLACYSLLV TVAFIIFWVR SKRSRLLHSD YMNMTPRRPGPTRKHYQPYA PPRDFAAYRS KKVAKKPTNK APHPKQEPQE INFPDDLPGSNTAAPVQETL HGCQPVTQED GKESRISVQE RQ 252 OSM_M912_MGVLLTQRTL LSLVLALLFP SMASMQVQLQ ESGPGLVKPS ETLSLTCTVS spCD8GGSVSSGSYY WSWIRQPPGK GLEWIGYIYY SGSTNYNPSL KSRVTISVDT _CD28_SKNQFSLKLS SVTAADTAVY YCAREGKNGA FDIWGQGTMV TVSSGGGGSG CD40GGGSGGGGSD IQMTQSPSSL SASVGDRVTI TCRASQSISS YLNWYQQKPG CTP239KAPKLLIYAA SSLQSGVPSG FSGSGSGTDF TLTISSLQPE DFATYYCQQSYSTPLTFGGG TKVEIKAAAG SGGSGFVPVF LPAKPTTTPA PRPPTPAPTIASQPLSLRPE ACRPAAGGAV HTRGLDFACD IYIWAPLAGT CGVLLLSLVITLYCNHRNRS KRSRLLHSDY MNMTPRRPGP TRKHYQPYAP PRDFAAYRSKKVAKKPTNKA PHPKQEPQEI NFPDDLPGSN TAAPVQETLH GCQPVTQEDG KESRISVQER Q 253CD8a_M912 MALPVTALLL PLALLLHAAR PQVQLQESGP GLVKPSETLS LTCTVSGGSV _spCD8SSGSYYWSWI RQPPGKGLEW IGYIYYSGST NYNPSLKSRV TISVDTSKNQ _CD28_CD4FSLKLSSVTA ADTAVYYCAR EGKNGAFDIW GQGTMVTVSS GGGGSGGGGS 0GGGGSDIQMT QSPSSLSASV GDRVTITCRA SQSISSYLNW YQQKPGKAPKLLIYAASSLQ SGVPSGFSGS GSGTDFTLTI SSLQPEDFAT YYCQQSYSTPLTFGGGTKVE IKAAAGSGGS GFVPVFLPAK PTTTPAPRPP TPAPTIASQPLSLRPEACRP AAGGAVHTRG LDFACDIYIW APLAGTCGVL LLSLVITLYCNHRNRSKRSR LLHSDYMNMT PRRPGPTRKH YQPYAPPRDF AAYRSKKVAKKPTNKAPHPK QEPQEINFPD DLPGSNTAAP VQETLHGCQP VTQEDGKESR ISVQERQ 254CD2_M912_ MSFPCKFVAS FLLIFNVSSK GAVSQVQLQE SGPGLVKPSE TLSLTCTVSG spCD8GSVSSGSYYW SWIRQPPGKG LEWIGYIYYS GSTNYNPSLK SRVTISVDTS _CD28_KNQFSLKLSS VTAADTAVYY CAREGKNGAF DIWGQGTMVT VSSGGGGSGG CD40GGSGGGGSDI QMTQSPSSLS ASVGDRVTIT CRASQSISSY LNWYQQKPGKAPKLLIYAAS SLQSGVPSGF SGSGSGTDFT LTISSLQPED FATYYCQQSYSTPLTFGGGT KVEIKAAAGS GGSGFVPVFL PAKPTTTPAP RPPTPAPTIASQPLSLRPEA CRPAAGGAVH TRGLDFACDI YIWAPLAGTC GVLLLSLVITLYCNHRNRSK RSRLLHSDYM NMTPRRPGPT RKHYQPYAPP RDFAAYRSKKVAKKPTNKAP HPKQEPQEIN FPDDLPGSNT AAPVQETLHG CQPVTQEDGK ESRISVQERQ 255IL2_M912_ MYRMQLLSCI ALSLALVTNS QVQLQESGPG LVKPSETLSL TCTVSGGSVS spCD8SGSYYWSWIR QPPGKGLEWI GYIYYSGSTN YNPSLKSRVT ISVDTSKNQF _CD28_SLKLSSVTAA DTAVYYCARE GKNGAFDIWG QGTMVTVSSG GGGSGGGGSG CD40GGGSDIQMTQ SPSSLSASVG DRVTITCRAS QSISSYLNWY QQKPGKAPKLLIYAASSLQS GVPSGFSGSG SGTDFTLTIS SLQPEDFATY YCQQSYSTPLTFGGGTKVEI KAAAGSGGSG FVPVFLPAKP TTTPAPRPPT PAPTIASQPLSLRPEACRPA AGGAVHTRGL DFACDIYIWA PLAGTCGVLL LSLVITLYCNHRNRSKRSRL LHSDYMNMTP RRPGPTRKHY QPYAPPRDFA AYRSKKVAKKPTNKAPHPKQ EPQEINFPDD LPGSNTAAPV QETLHGCQPV TQEDGKESRI SVQERQ 256 GM-CSFMWLQSLLLLG TVACSISQVQ LQESGPGLVK PSETLSLTCT VSGGSVSSGS _M912_spCYYWSWIRQPP GKGLEWIGYI YYSGSTNYNP SLKSRVTISV DTSKNQFSLK D8LSSVTAADTA VYYCAREGKN GAFDIWGQGT MVTVSSGGGG SGGGGSGGGG _CD28_SDIQMTQSPS SLSASVGDRV TITCRASQSI SSYLNWYQQK PGKAPKLLIY CD40AASSLQSGVP SGFSGSGSGT DFTLTISSLQ PEDFATYYCQ QSYSTPLTFGGGTKVEIKAA AGSGGSGFVP VFLPAKPTTT PAPRPPTPAP TIASQPLSLRPEACRPAAGG AVHTRGLDFA CDIYIWAPLA GTCGVLLLSL VITLYCNHRNRSKRSRLLHS DYMNMTPRRP GPTRKHYQPY APPRDFAAYR SKKVAKKPTNKAPHPKQEPQ EINFPDDLPG SNTAAPVQET LHGCQPVTQE DGKESRISVQ ERQ 257 hIgGk-MEAPAQLLFL LLLWLPDTTR QVQLQESGPG LVKPSETLSL TCTVSGGSVS VIIISGSYYWSWIR QPPGKGLEWI GYIYYSGSTN YNPSLKSRVT ISVDTSKNQF _M912_spCSLKLSSVTAA DTAVYYCARE GKNGAFDIWG QGTMVTVSSG GGGSGGGGSG D8GGGSDIQMTQ SPSSLSASVG DRVTITCRAS QSISSYLNWY QQKPGKAPKL _CD28_LIYAASSLQS GVPSGFSGSG SGTDFTLTIS SLQPEDFATY YCQQSYSTPL CD40TFGGGTKVEI KAAAGSGGSG FVPVFLPAKP TTTPAPRPPT PAPTIASQPLSLRPEACRPA AGGAVHTRGL DFACDIYIWA PLAGTCGVLL LSLVITLYCNHRNRSKRSRL LHSDYMNMTP RRPGPTRKHY QPYAPPRDFA AYRSKKVAKKPTNKAPHPKQ EPQEINFPDD LPGSNTAAPV QETLHGCQPV TQEDGKESRI SVQERQ 258OSM_M912 MGVLLTQRTL LSLVLALLFP SMASMQVQLQ ESGPGLVKPS ETLSLTCTVS _CD28TMGGSVSSGSYY WSWIRQPPGK GLEWIGYIYY SGSTNYNPSL KSRVTISVDT _CD28_SKNQFSLKLS SVTAADTAVY YCAREGKNGA FDIWGQGTMV TVSSGGGGSG CD40GGGSGGGGSD IQMTQSPSSL SASVGDRVTI TCRASQSISS YLNWYQQKPGKAPKLLIYAA SSLQSGVPSG FSGSGSGTDF TLTISSLQPE DFATYYCQQSYSTPLTFGGG TKVEIKAAAG SGGSGFWVLV VVGGVLACYS LLVTVAFIIFWVRSKRSRLL HSDYMNMTPR RPGPTRKHYQ PYAPPRDFAA YRSKKVAKKPTNKAPHPKQE PQEINFPDDL PGSNTAAPVQ ETLHGCQPVT QEDGKESRIS VQERQ 259CD8a_M912 MALPVTALLL PLALLLHAAR PQVQLQESGP GLVKPSETLS LTCTVSGGSV _CD28TMSSGSYYWSWI RQPPGKGLEW IGYIYYSGST NYNPSLKSRV TISVDTSKNQ _CD28_CD4FSLKLSSVTA ADTAVYYCAR EGKNGAFDIW GQGTMVTVSS GGGGSGGGGS 0GGGGSDIQMT QSPSSLSASV GDRVTITCRA SQSISSYLNW YQQKPGKAPKLLIYAASSLQ SGVPSGFSGS GSGTDFTLTI SSLQPEDFAT YYCQQSYSTPLTFGGGTKVE IKAAAGSGGS GFWVLVVVGG VLACYSLLVT VAFIIFWVRSKRSRLLHSDY MNMTPRRPGP TRKHYQPYAP PRDFAAYRSK KVAKKPTNKAPHPKQEPQEI NFPDDLPGSN TAAPVQETLH GCQPVTQEDG KESRISVQER Q 260 CD2_M912MSFPCKFVAS FLLIFNVSSK GAVSQVQLQE SGPGLVKPSE TLSLTCTVSG _CD28TMGSVSSGSYYW SWIRQPPGKG LEWIGYIYYS GSTNYNPSLK SRVTISVDTS _CD28_KNQFSLKLSS VTAADTAVYY CAREGKNGAF DIWGQGTMVT VSSGGGGSGG CD40GGSGGGGSDI QMTQSPSSLS ASVGDRVTIT CRASQSISSY LNWYQQKPGKAPKLLIYAAS SLQSGVPSGF SGSGSGTDFT LTISSLQPED FATYYCQQSYSTPLTFGGGT KVEIKAAAGS GGSGFWVLVV VGGVLACYSL LVTVAFIIFWVRSKRSRLLH SDYMNMTPRR PGPTRKHYQP YAPPRDFAAY RSKKVAKKPTNKAPHPKQEP QEINFPDDLP GSNTAAPVQE TLHGCQPVTQ EDGKESRISV QERQ 261 IL2_M912MYRMQLLSCI ALSLALVTNS QVQLQESGPG LVKPSETLSL TCTVSGGSVS _CD28TMSGSYYWSWIR QPPGKGLEWI GYIYYSGSTN YNPSLKSRVT ISVDTSKNQF _CD28_SLKLSSVTAA DTAVYYCARE GKNGAFDIWG QGTMVTVSSG GGGSGGGGSG CD40GGGSDIQMTQ SPSSLSASVG DRVTITCRAS QSISSYLNWY QQKPGKAPKLLIYAASSLQS GVPSGFSGSG SGTDFTLTIS SLQPEDFATY YCQQSYSTPLTFGGGTKVEI KAAAGSGGSG FWVLVVVGGV LACYSLLVTV AFIIFWVRSKRSRLLHSDYM NMTPRRPGPT RKHYQPYAPP RDFAAYRSKK VAKKPTNKAPHPKQEPQEIN FPDDLPGSNT AAPVQETLHG CQPVTQEDGK ESRISVQERQ 262 GM-MWLQSLLLLG TVACSISQVQ LQESGPGLVK PSETLSLTCT VSGGSVSSGS CSF_M912YYWSWIRQPP GKGLEWIGYI YYSGSTNYNP SLKSRVTISV DTSKNQFSLK _CD28TMLSSVTAADTA VYYCAREGKN GAFDIWGQGT MVTVSSGGGG SGGGGSGGGG _CD28_SDIQMTQSPS SLSASVGDRV TITCRASQSI SSYLNWYQQK PGKAPKLLIY CD40AASSLQSGVP SGFSGSGSGT DFTLTISSLQ PEDFATYYCQ QSYSTPLTFGGGTKVEIKAA AGSGGSGFWV LVVVGGVLAC YSLLVTVAFI IFWVRSKRSRLLHSDYMNMT PRRPGPTRKH YQPYAPPRDF AAYRSKKVAK KPTNKAPHPKQEPQEINFPD DLPGSNTAAP VQETLHGCQP VTQEDGKESR ISVQERQ 263 hIgGk-MEAPAQLLFL LLLWLPDTTR QVQLQESGPG LVKPSETLSL TCTVSGGSVS VIIISGSYYWSWIR QPPGKGLEWI GYIYYSGSTN YNPSLKSRVT ISVDTSKNQF _M912SLKLSSVTAA DTAVYYCARE GKNGAFDIWG QGTMVTVSSG GGGSGGGGSG _CD28TMGGGSDIQMTQ SPSSLSASVG DRVTITCRAS QSISSYLNWY QQKPGKAPKL _CD28_LIYAASSLQS GVPSGFSGSG SGTDFTLTIS SLQPEDFATY YCQQSYSTPL CD40TFGGGTKVEI KAAAGSGGSG FWVLVVVGGV LACYSLLVTV AFIIFWVRSKRSRLLHSDYM NMTPRRPGPT RKHYQPYAPP RDFAAYRSKK VAKKPTNKAPHPKQEPQEIN FPDDLPGSNT AAPVQETLHG CQPVTQEDGK ESRISVQERQ 264 OSM_HuYP2MGVLLTQRTL LSLVLALLFP SMASMEVQLV ESGGGLVQPG GSLRLSCAAS 18GFDLGFYFYA CWVRQAPGKG LEWVSCIYTA GSGSTYYASW AKGRFTISRD _spCD28NSKNTLYLQM NSLRAEDTAV YYCARSTANT RSTYYLNLWG QGTLVTVSSG _CD28_GGGSGGGGSG GGGSDIQMTQ SPSSLSASVG DRVTITCQAS QRISSYLSWY CD40QQKPGKVPKL LIYGASTLAS GVPSRFSGSG SGTDFTLTIS SLQPEDVATY CTP228YCQSYAYFDS NNWHAFGGGT KVEIAAAGSG GSGILVKQSP MLVAYDNAVNLSCKYSYNLF SREFRASLHK GLDSAVEVCV VYGNYSQQLQ VYSKTGFNCDGKLGNESVTF YLQNLYVNQT DIYFCKIEVM YPPPYLDNEK SNGTIIHVKGKHLCPSPLFP GPSKPFWVLV VVGGVLACYS LLVTVAFIIF WVRSKRSRLLHSDYMNMTPR RPGPTRKHYQ PYAPPRDFAA YRSKKVAKKP TNKAPHPKQEPQEINFPDDL PGSNTAAPVQ ETLHGCQPVT QEDGKESRIS VQERQ 265 CD8a_HuYPMALPVTALLL PLALLLHAAR PEVQLVESGG GLVQPGGSLR LSCAASGFDL 218GFYFYACWVR QAPGKGLEWV SCIYTAGSGS TYYASWAKGR FTISRDNSKN _spCD28TLYLQMNSLR AEDTAVYYCA RSTANTRSTY YLNLWGQGTL VTVSSGGGGS _CD28_GGGGSGGGGS DIQMTQSPSS LSASVGDRVT ITCQASQRIS SYLSWYQQKP CD40GKVPKLLIYG ASTLASGVPS RFSGSGSGTD FTLTISSLQP EDVATYYCQSYAYFDSNNWH AFGGGTKVEI AAAGSGGSGI LVKQSPMLVA YDNAVNLSCKYSYNLFSREF RASLHKGLDS AVEVCVVYGN YSQQLQVYSK TGFNCDGKLGNESVTFYLQN LYVNQTDIYF CKIEVMYPPP YLDNEKSNGT IIHVKGKHLCPSPLFPGPSK PFWVLVVVGG VLACYSLLVT VAFIIFWVRS KRSRLLHSDYMNMTPRRPGP TRKHYQPYAP PRDFAAYRSK KVAKKPTNKA PHPKQEPQEINFPDDLPGSN TAAPVQETLH GCQPVTQEDG KESRISVQER Q 266 CD2_HuYP2MSFPCKFVAS FLLIFNVSSK GAVSEVQLVE SGGGLVQPGG SLRLSCAASG 18FDLGFYFYAC WVRQAPGKGL EWVSCIYTAG SGSTYYASWA KGRFTISRDN _spCD28SKNTLYLQMN SLRAEDTAVY YCARSTANTR STYYLNLWGQ GTLVTVSSGG _CD28_GGSGGGGSGG GGSDIQMTQS PSSLSASVGD RVTITCQASQ RISSYLSWYQ CD40QKPGKVPKLL IYGASTLASG VPSRFSGSGS GTDFTLTISS LQPEDVATYYCQSYAYFDSN NWHAFGGGTK VEIAAAGSGG SGILVKQSPM LVAYDNAVNLSCKYSYNLFS REFRASLHKG LDSAVEVCVV YGNYSQQLQV YSKTGFNCDGKLGNESVTFY LQNLYVNQTD IYFCKIEVMY PPPYLDNEKS NGTIIHVKGKHLCPSPLFPG PSKPFWVLVV VGGVLACYSL LVTVAFIIFW VRSKRSRLLHSDYMNMTPRR PGPTRKHYQP YAPPRDFAAY RSKKVAKKPT NKAPHPKQEPQEINFPDDLP GSNTAAPVQE TLHGCQPVTQ EDGKESRISV QERQ 267 IL2_HuYP2MYRMQLLSCI ALSLALVTNS EVQLVESGGG LVQPGGSLRL SCAASGFDLG 18FYFYACWVRQ APGKGLEWVS GIYTAGSGST YYASWAKGRF TISRDNSKNT _spCD28LYLQMNSLRA EDTAVYYCAR STANTRSTYY LNLWGQGTLV TVSSGGGGSG _CD28_GGGSGGGGSD IQMTQSPSSL SASVGDRVTI TCQASQRISS YLSWYQQKPG CD40KVPKLLIYGA STLASGVPSR FSGSGSGTDF TLTISSLQPE DVATYYCQSYAYFDSNNWHA FGGGTKVEIA AAGSGGSGIL VKQSPMLVAY DNAVNLSCKYSYNLFSREFR ASLHKGLDSA VEVCVVYGNY SQQLQVYSKT GFNCDGKLGNESVTFYLQNL YVNQTDIYFC KIEVMYPPPY LDNEKSNGTI IHVKGKHLCPSPLFPGPSKP FWVLVVVGGV LACYSLLVTV AFIIFWVRSK RSRLLHSDYMNMTPRRPGPT RKHYQPYAPP RDFAAYRSKK VAKKPTNKAP HPKQEPQEINFPDDLPGSNT AAPVQETLHG CQPVTQEDGK ESRISVQERQ 268 GM-MWLQSLLLLG TVACSISEVQ LVESGGGLVQ PGGSLRLSCA ASGFDLGFYF CSF_HuYP2YACWVRQAPG KGLEWVSCIY TAGSGSTYYA SWAKGRFTIS RDNSKNTLYL 18QMNSLRAEDT AVYYCARSTA NTRSTYYLNL WGQGTLVTVS SGGGGSGGGG _spCD28SGGGGSDIQM TQSPSSLSAS VGDRVTITCQ ASQRISSYLS WYQQKPGKVP _CD28_KLLIYGASTL ASGVPSRFSG SGSGTDFTLT ISSLQPEDVA TYYCQSYAYF CD40DSNNWHAFGG GTKVEIAAAG SGGSGILVKQ SPMLVAYDNA VNLSCKYSYNLFSREFRASL HKGLDSAVEV CVVYGNYSQQ LQVYSKTGFN CDGKLGNESVTFYLQNLYVN QTDIYFCKIE VMYPPPYLDN EKSNGTIIHV KGKHLCPSPLFPGPSKPFWV LVVVGGVLAC YSLLVTVAFI IFWVRSKRSR LLHSDYMNMTPRRPGPTRKH YQPYAPPRDF AAYRSKKVAK KPTNKAPHPK QEPQEINFPDDLPGSNTAAP VQETLHGCQP VTQEDGKESR ISVQERQ 269 hIgGk-MEAPAQLLFL LLLWLPDTTR EVQLVESGGG LVQPGGSLRL SCAASGFDLG VIIIFYFYACWVRQ APGKGLEWVS GIYTAGSGST YYASWAKGRF TISRDNSKNT _HuYP218LYLQMNSLRA EDTAVYYCAR STANTRSTYY LNLWGQGTLV TVSSGGGGSG _spCD28GGGSGGGGSD IQMTQSPSSL SASVGDRVTI TCQASQRISS YLSWYQQKPG _CD28_KVPKLLIYGA STLASGVPSR FSGSGSGTDF TLTISSLQPE DVATYYCQSY CD40AYFDSNNWHA FGGGTKVEIA AAGSGGSGIL VKQSPMLVAY DNAVNLSCKYSYNLFSREFR ASLHKGLDSA VEVCVVYGNY SQQLQVYSKT GFNCDGKLGNESVTFYLQNL YVNQTDIYFC KIEVMYPPPY LDNEKSNGTI IHVKGKHLCPSPLFPGPSKP FWVLVVVGGV LACYSLLVTV AFIIFWVRSK RSRLLHSDYMNMTPRRPGPT RKHYQPYAPP RDFAAYRSKK VAKKPTNKAP HPKQEPQEINFPDDLPGSNT AAPVQETLHG CQPVTQEDGK ESRISVQERQ 270 OSM_HuYP2MGVLLTQRTL LSLVLALLFP SMASMEVQLV ESGGGLVQPG GSLRLSCAAS 18GFDLGFYFYA CWVRQAPGKG LEWVSCIYTA GSGSTYYASW AKGRFTISRD _spCD8NSKNTLYLQM NSLRAEDTAV YYCARSTANT RSTYYLNLWG QGTLVTVSSG _CD28_GGGSGGGGSG GGGSDIQMTQ SPSSLSASVG DRVTITCQAS QRISSYLSWY CD40QQKPGKVPKL LIYGASTLAS GVPSRFSGSG SGTDFTLTIS SLQPEDVATY CTP240YCQSYAYFDS NNWHAFGGGT KVEIAAAGSG GSGFVPVFLP AKPTTTPAPRPPTPAPTIAS QPLSLRPEAC RPAAGGAVHT RGLDFACDIY IWAPLAGTCGVLLLSLVITL YCNHRNRSKR SRLLHSDYMN MTPRRPGPTR KHYQPYAPPRDFAAYRSKKV AKKPTNKAPH PKQEPQEINF PDDLPGSNTA APVQETLHGCQPVTQEDGKE SRISVQERQ 271 CD8a_HuYPMALPVTALLL PLALLLHAAR PEVQLVESGG GLVQPGGSLR LSCAASGFDL 218GFYFYACWVR QAPGKGLEWV SCIYTAGSGS TYYASWAKGR FTISRDNSKN _spCD8TLYLQMNSLR AEDTAVYYCA RSTANTRSTY YLNLWGQGTL VTVSSGGGGS _CD28_GGGGSGGGGS DIQMTQSPSS LSASVGDRVT ITCQASQRIS SYLSWYQQKP CD40GKVPKLLIYG ASTLASGVPS RFSGSGSGTD FTLTISSLQP EDVATYYCQSYAYFDSNNWH AFGGGTKVEI AAAGSGGSGF VPVFLPAKPT TTPAPRPPTPAPTIASQPLS LRPEACRPAA GGAVHTRGLD FACDIYIWAP LAGTCGVLLLSLVITLYCNH RNRSKRSRLL HSDYMNMTPR RPGPTRKHYQ PYAPPRDFAAYRSKKVAKKP TNKAPHPKQE PQEINFPDDL PGSNTAAPVQ ETLHGCQPVT QEDGKESRIS VQERQ272 CD2_HuYP2 MSFPCKFVAS FLLIFNVSSK GAVSEVQLVE SGGGLVQPGG SLRLSCAASG 18FDLGFYFYAC WVRQAPGKGL EWVSCIYTAG SGSTYYASWA KGRFTISRDN _spCD8SKNTLYLQMN SLRAEDTAVY YCARSTANTR STYYLNLWGQ GTLVTVSSGG _CD28_CD4GGSGGGGSGG GGSDIQMTQS PSSLSASVGD RVTITCQASQ RISSYLSWYQ 0QKPGKVPKLL IYGASTLASG VPSRFSGSGS GTDFTLTISS LQPEDVATYYCQSYAYFDSN NWHAFGGGTK VEIAAAGSGG SGFVPVFLPA KPTTTPAPRPPTPAPTIASQ PLSLRPEACR PAAGGAVHTR GLDFACDIYI WAPLAGTCGVLLLSLVITLY CNHRNRSKRS RLLHSDYMNM TPRRPGPTRK HYQPYAPPRDFAAYRSKKVA KKPTNKAPHP KQEPQEINFP DDLPGSNTAA PVQETLHGCQPVTQEDGKES RISVQERQ 273 IL2_HuYP2MYRMQLLSCI ALSLALVTNS EVQLVESGGG LVQPGGSLRL SCAASGFDLG 18FYFYACWVRQ APGKGLEWVS GIYTAGSGST YYASWAKGRF TISRDNSKNT _spCD8LYLQMNSLRA EDTAVYYCAR STANTRSTYY LNLWGQGTLV TVSSGGGGSG _CD28_CD4GGGSGGGGSD IQMTQSPSSL SASVGDRVTI TCQASQRISS YLSWYQQKPG 0KVPKLLIYGA STLASGVPSR FSGSGSGTDF TLTISSLQPE DVATYYCQSYAYFDSNNWHA FGGGTKVEIA AAGSGGSGFV PVFLPAKPTT TPAPRPPTPAPTIASQPLSL RPEACRPAAG GAVHTRGLDF ACDIYIWAPL AGTCGVLLLSLVITLYCNHR NRSKRSRLLH SDYMNMTPRR PGPTRKHYQP YAPPRDFAAYRSKKVAKKPT NKAPHPKQEP QEINFPDDLP GSNTAAPVQE TLHGCQPVTQ EDGKESRISV QERQ274 GM-CSF MWLQSLLLLG TVACSISEVQ LVESGGGLVQ PGGSLRLSCA ASGFDLGFYF_HuYP218 YACWVRQAPG KGLEWVSCIY TAGSGSTYYA SWAKGRFTIS RDNSKNTLYL _spCD8QMNSLRAEDT AVYYCARSTA NTRSTYYLNL WGQGTLVTVS SGGGGSGGGG _CD28_CD4SGGGGSDIQM TQSPSSLSAS VGDRVTITCQ ASQRISSYLS WYQQKPGKVP 0KLLIYGASTL ASGVPSRFSG SGSGTDFTLT ISSLQPEDVA TYYCQSYAYFDSNNWHAFGG GTKVEIAAAG SGGSGFVPVF LPAKPTTTPA PRPPTPAPTIASQPLSLRPE ACRPAAGGAV HTRGLDFACD IYIWAPLAGT CGVLLLSLVITLYCNHRNRS KRSRLLHSDY MNMTPRRPGP TRKHYQPYAP PRDFAAYRSKKVAKKPTNKA PHPKQEPQEI NFPDDLPGSN TAAPVQETLH GCQPVTQEDG KESRISVQER Q 275hIgGk- MEAPAQLLFL LLLWLPDTTR EVQLVESGGG LVQPGGSLRL SCAASGFDLG VIIIFYFYACWVRQ APGKGLEWVS GIYTAGSGST YYASWAKGRF TISRDNSKNT _HuYP218LYLQMNSLRA EDTAVYYCAR STANTRSTYY LNLWGQGTLV TVSSGGGGSG _spCD8GGGSGGGGSD IQMTQSPSSL SASVGDRVTI TCQASQRISS YLSWYQQKPG _CD28_CD4KVPKLLIYGA STLASGVPSR FSGSGSGTDF TLTISSLQPE DVATYYCQSY 0AYFDSNNWHA FGGGTKVEIA AAGSGGSGFV PVFLPAKPTT TPAPRPPTPAPTIASQPLSL RPEACRPAAG GAVHTRGLDF ACDIYIWAPL AGTCGVLLLSLVITLYCNHR NRSKRSRLLH SDYMNMTPRR PGPTRKHYQP YAPPRDFAAYRSKKVAKKPT NKAPHPKQEP QEINFPDDLP GSNTAAPVQE TLHGCQPVTQ EDGKESRISV QERQ276 OSM_HuYP2 MGVLLTQRTL LSLVLALLFP SMASMEVQLV ESGGGLVQPG GSLRLSCAAS 18GFDLGFYFYA CWVRQAPGKG LEWVSCIYTA GSGSTYYASW AKGRFTISRD _CD28TMNSKNTLYLQM NSLRAEDTAV YYCARSTANT RSTYYLNLWG QGTLVTVSSG _CD28_CD4GGGSGGGGSG GGGSDIQMTQ SPSSLSASVG DRVTITCQAS QRISSYLSWY 0QQKPGKVPKL LIYGASTLAS GVPSRFSGSG SGTDFTLTIS SLQPEDVATYYCQSYAYFDS NNWHAFGGGT KVEIAAAGSG GSGFWVLVVV GGVLACYSLLVTVAFIIFWV RSKRSRLLHS DYMNMTPRRP GPTRKHYQPY APPRDFAAYRSKKVAKKPTN KAPHPKQEPQ EINFPDDLPG SNTAAPVQET LHGCQPVTQE DGKESRISVQ ERQ277 CD8a_HuYP MALPVTALLL PLALLLHAAR PEVQLVESGG GLVQPGGSLR LSCAASGFDL 218GFYFYACWVR QAPGKGLEWV SCIYTAGSGS TYYASWAKGR FTISRDNSKN _CD28TMTLYLQMNSLR AEDTAVYYCA RSTANTRSTY YLNLWGQGTL VTVSSGGGGS _CD28_GGGGSGGGGS DIQMTQSPSS LSASVGDRVT ITCQASQRIS SYLSWYQQKP CD40GKVPKLLIYG ASTLASGVPS RFSGSGSGTD FTLTISSLQP EDVATYYCQSYAYFDSNNWH AFGGGTKVEI AAAGSGGSGF WVLVVVGGVL ACYSLLVTVAFIIFWVRSKR SRLLHSDYMN MTPRRPGPTR KHYQPYAPPR DFAAYRSKKVAKKPTNKAPH PKQEPQEINF PDDLPGSNTA APVQETLHGC QPVTQEDGKE SRISVQERQ 278CD2_HuYP2 MSFPCKFVAS FLLIFNVSSK GAVSEVQLVE SGGGLVQPGG SLRLSCAASG 18FDLGFYFYAC WVRQAPGKGL EWVSCIYTAG SGSTYYASWA KGRFTISRDN _CD28TMSKNTLYLQMN SLRAEDTAVY YCARSTANTR STYYLNLWGQ GTLVTVSSGG _CD28_GGSGGGGSGG GGSDIQMTQS PSSLSASVGD RVTITCQASQ RISSYLSWYQ CD40QKPGKVPKLL IYGASTLASG VPSRFSGSGS GTDFTLTISS LQPEDVATYYCQSYAYFDSN NWHAFGGGTK VEIAAAGSGG SGFWVLVVVG GVLACYSLLVTVAFIIFWVR SKRSRLLHSD YMNMTPRRPG PTRKHYQPYA PPRDFAAYRSKKVAKKPTNK APHPKQEPQE INFPDDLPGS NTAAPVQETL HGCQPVTQED GKESRISVQE RQ 279IL2_HuYP2 MYRMQLLSCI ALSLALVTNS EVQLVESGGG LVQPGGSLRL SCAASGFDLG 18FYFYACWVRQ APGKGLEWVS GIYTAGSGST YYASWAKGRF TISRDNSKNT _CD28TMLYLQMNSLRA EDTAVYYCAR STANTRSTYY LNLWGQGTLV TVSSGGGGSG _CD28_GGGSGGGGSD IQMTQSPSSL SASVGDRVTI TCQASQRISS YLSWYQQKPG CD40KVPKLLIYGA STLASGVPSR FSGSGSGTDF TLTISSLQPE DVATYYCQSYAYFDSNNWHA FGGGTKVEIA AAGSGGSGFW VLVVVGGVLA CYSLLVTVAFIIFWVRSKRS RLLHSDYMNM TPRRPGPTRK HYQPYAPPRD FAAYRSKKVAKKPTNKAPHP KQEPQEINFP DDLPGSNTAA PVQETLHGCQ PVTQEDGKES RISVQERQ 280 GM-MWLQSLLLLG TVACSISEVQ LVESGGGLVQ PGGSLRLSCA ASGFDLGFYF CSF_HuYP2YACWVRQAPG KGLEWVSCIY TAGSGSTYYA SWAKGRFTIS RDNSKNTLYL 18QMNSLRAEDT AVYYCARSTA NTRSTYYLNL WGQGTLVTVS SGGGGSGGGG _CD28TMSGGGGSDIQM TQSPSSLSAS VGDRVTITCQ ASQRISSYLS WYQQKPGKVP _CD28_KLLIYGASTL ASGVPSRFSG SGSGTDFTLT ISSLQPEDVA TYYCQSYAYF CD40DSNNWHAFGG GTKVEIAAAG SGGSGFWVLV VVGGVLACYS LLVTVAFIIFWVRSKRSRLL HSDYMNMTPR RPGPTRKHYQ PYAPPRDFAA YRSKKVAKKPTNKAPHPKQE PQEINFPDDL PGSNTAAPVQ ETLHGCQPVT QEDGKESRIS VQERQ 281 hIgGk-MEAPAQLLFL LLLWLPDTTR EVQLVESGGG LVQPGGSLRL SCAASGFDLG VIIIFYFYACWVRQ APGKGLEWVS GIYTAGSGST YYASWAKGRF TISRDNSKNT _HuYP218LYLQMNSLRA EDTAVYYCAR STANTRSTYY LNLWGQGTLV TVSSGGGGSG _CD28TMGGGSGGGGSD IQMTQSPSSL SASVGDRVTI TCQASQRISS YLSWYQQKPG _CD28_KVPKLLIYGA STLASGVPSR FSGSGSGTDF TLTISSLQPE DVATYYCQSY CD40AYFDSNNWHA FGGGTKVEIA AAGSGGSGFW VLVVVGGVLA CYSLLVTVAFIIFWVRSKRS RLLHSDYMNM TPRRPGPTRK HYQPYAPPRD FAAYRSKKVAKKPTNKAPHP KQEPQEINFP DDLPGSNTAA PVQETLHGCQ PVTQEDGKES RISVQERQ 282OSM_P4 MGVLLTQRTL LSLVLALLFP SMASMQVOLQ QSGPGLVTPS QTLSLTCAIS _spCD28GDSVSSNSAT WNWIRQSPSR GLEWLGRTYY RSKWYNDYAV SVKSRMSINP _CD28_DTSKNQFSLQ LNSVTPEDTA VYYCARGMMT YYYGMDVWGQ GTTVTVSSGG CD40GGSGGGGSGG GGSQPVLTQS SSLSASPGAS ASLTCTLRSG INVGPYRIYW CTP229YQQKPGSPPQ YLLNYKSDSD KQQGSGVPSR FSGSKDASAN AGVLLISGLRSEDEADYYGM IWHSSAAVFG GGTQLTVLSA AAGSGGSGIL VKQSPMLVAYDNAVNLSCKY SYNLFSREFR ASLHKGLDSA VEVCVVYGNY SQQLQVYSKTGFNCDGKLGN ESVTFYLQNL YVNQTDIYFC KIEVMYPPPY LDNEKSNGTIIHVKGKHLCP SPLFPGPSKP FWVLVVVGGV LACYSLLVTV AFIIFWVRSKRSRLLHSDYM NMTPRRPGPT RKHYQPYAPP RDFAAYRSKK VAKKPTNKAPHPKQEPQEIN FPDDLPGSNT AAPVQETLHG CQPVTQEDGK ESRISVQERQ 283 CD8a_P4MALPVTALLL PLALLLHAAR PQVQLQQSGP GLVTPSQTLS LTCAISGDSV _spCD28SSNSATWNWI RQSPSRGLEW LGRTYYRSKW YNDYAVSVKS RMSINPDTSK _CD28_NQFSLQLNSV TPEDTAVYYC ARGMMTYYYG MDVWGQGTTV TVSSGGGGSG CD40GGGSGGGGSQ PVLTQSSSLS ASPGASASLT CTLRSGINVG PYRIYWYQQKPGSPPQYLLN YKSDSDKQQG SGVPSRFSGS KDASANAGVL LISGLRSEDEADYYCMIWHS SAAVFGGGTQ LTVLSAAAGS GGSGILVKQS PMLVAYDNAVNLSCKYSYNL FSREFRASLH KGLDSAVEVC VVYGNYSQQL QVYSKTGFNCDGKLGNESVT FYLQNLYVNQ TDIYFCKIEV MYPPPYLDNE KSNGTIIHVKGKHLCPSPLF PGPSKPFWVL VVVGGVLACY SLLVTVAFII FWVRSKRSRLLHSDYMNMTP RRPGPTRKHY QPYAPPRDFA AYRSKKVAKK PTNKAPHPKQEPQEINFPDD LPGSNTAAPV QETLHGCQPV TQEDGKESRI SVQERQ 284 CD2_P4MSFPCKFVAS FLLIFNVSSK GAVSQVQLQQ SGPGLVTPSQ TLSLTCAISG _spCD28DSVSSNSATW NWIRQSPSRG LEWLGRTYYR SKWYNDYAVS VKSRMSINPD _CD28_TSKNQFSLQL NSVTPEDTAV YYCARGMMTY YYGMDVWGQG TTVTVSSGGG CD40GSGGGGSGGG GSQPVLTQSS SLSASPGASA SLTCTLRSGI NVGPYRIYWYQQKPGSPPQY LLNYKSDSDK QQGSGVPSRF SGSKDASANA GVLLISGLRSEDEADYYCMI WHSSAAVFGG GTQLTVLSAA AGSGGSGILV KQSPMLVAYDNAVNLSCKYS YNLFSREFRA SLHKGLDSAV EVCVVYGNYS QQLQVYSKTGFNCDGKLGNE SVTFYLQNLY VNQTDIYFCK IEVMYPPPYL DNEKSNGTIIHVKGKHLCPS PLFPGPSKPF WVLVVVGGVL ACYSLLVTVA FIIFWVRSKRSRLLHSDYMN MTPRRPGPTR KHYQPYAPPR DFAAYRSKKV AKKPTNKAPHPKQEPQEINF PDDLPGSNTA APVQETLHGC QPVTQEDGKE SRISVQERQ 285 IL2_P4MYRMQLLSCI ALSLALVTNS QVQLQQSGPG LVTPSQTLSL TCAISGDSVS _spCD28SNSATWNWIR QSPSRGLEWL GRTYYRSKWY NDYAVSVKSR MSINPDTSKN _CD28_QFSLQLNSVT PEDTAVYYCA RGMMTYYYGM DVWGQGTTVT VSSGGGGSGG CD40GGSGGGGSQP VLTQSSSLSA SPGASASLTC TLRSGINVGP YRIYWYQQKPGSPPQYLLNY KSDSDKQQGS GVPSRFSGSK DASANAGVLL ISGLRSEDEADYYCMIWHSS AAVFGGGTQL TVLSAAAGSG GSGILVKQSP MLVAYDNAVNLSCKYSYNLF SREFRASLHK GLDSAVEVCV VYGNYSQQLQ VYSKTGFNCDGKLGNESVTF YLQNLYVNQT DIYFCKIEVM YPPPYLDNEK SNGTIIHVKGKHLCPSPLFP GPSKPFWVLV VVGGVLACYS LLVTVAFIIF WVRSKRSRLLHSDYMNMTPR RPGPTRKHYQ PYAPPRDFAA YRSKKVAKKP TNKAPHPKQEPQEINFPDDL PGSNTAAPVQ ETLHGCQPVT QEDGKESRIS VQERQ 286 GM-CSF_P4MWLQSLLLLG TVACSISQVQ LQQSGPGLVT PSQTLSLTCA ISGDSVSSNS _spCD28ATWNWIRQSP SRGLEWLGRT YYRSKWYNDY AVSVKSRMSI NPDTSKNQFS _CD28_LQLNSVTPED TAVYYCARGM MTYYYGMDVW GQGTTVTVSS GGGGSGGGGS CD40GGGGSQPVLT QSSSLSASPG ASASLTCTLR SGINVGPYRI YWYQQKPGSPPQYLLNYKSD SDKQQGSGVP SRFSGSKDAS ANAGVLLISG LRSEDEADYYCMIWHSSAAV FGGGTQLTVL SAAAGSGGSG ILVKQSPMLV AYDNAVNLSCKYSYNLFSRE FRASLHKGLD SAVEVCVVYG NYSQQLQVYS KTGFNCDGKLGNESVTFYLQ NLYVNQTDIY FCKIEVMYPP PYLDNEKSNG TIIHVKGKHLCPSPLFPGPS KPFWVLVVVG GVLACYSLLV TVAFIIFWVR SKRSRLLHSDYMNMTPRRPG PTRKHYQPYA PPRDFAAYRS KKVAKKPTNK APHPKQEPQEINFPDDLPGS NTAAPVQETL HGCQPVTQED GKESRISVQE RQ 287 hIgGk-MEAPAQLLFL LLLWLPDTTR QVQLQQSGPG LVTPSQTLSL TCAISGDSVS VIIISNSATWNWIR QSPSRGLEWL GRTYYRSKWY NDYAVSVKSR MSINPDTSKN _P4QFSLQLNSVT PEDTAVYYCA RGMMTYYYGM DVWGQGTTVT VSSGGGGSGG _spCD28GGSGGGGSQP VLTQSSSLSA SPGASASLTC TLRSGINVGP YRIYWYQQKP _CD28_GSPPQYLLNY KSDSDKQQGS GVPSRFSGSK DASANAGVLL ISGLRSEDEA CD40DYYCMIWHSS AAVFGGGTQL TVLSAAAGSG GSGILVKQSP MLVAYDNAVNLSCKYSYNLF SREFRASLHK GLDSAVEVCV VYGNYSQQLQ VYSKTGFNCDGKLGNESVTF YLQNLYVNQT DIYFCKIEVM YPPPYLDNEK SNGTIIHVKGKHLCPSPLFP GPSKPFWVLV VVGGVLACYS LLVTVAFIIF WVRSKRSRLLHSDYMNMTPR RPGPTRKHYQ PYAPPRDFAA YRSKKVAKKP TNKAPHPKQEPQEINFPDDL PGSNTAAPVQ ETLHGCQPVT QEDGKESRIS VQERQ 288 OSM_P4_spMGVLLTQRTL LSLVLALLFP SMASMQVOLQ QSGPGLVTPS QTLSLTCAIS CD8GDSVSSNSAT WNWIRQSPSR GLEWLGRTYY RSKWYNDYAV SVKSRMSINP _CD28_DTSKNQFSLQ LNSVTPEDTA VYYCARGMMT YYYGMDVWGQ GTTVTVSSGG CD40GGSGGGGSGG GGSQPVLTQS SSLSASPGAS ASLTCTLRSG INVGPYRIYW CTP241YQQKPGSPPQ YLLNYKSDSD KQQGSGVPSR FSGSKDASAN AGVLLISGLRSEDEADYYCM IWHSSAAVFG GGTQLTVLSA AAGSGGSGFV PVFLPAKPTTTPAPRPPTPA PTIASQPLSL RPEACRPAAG GAVHTRGLDF ACDIYIWAPLAGTCGVLLLS LVITLYCNHR NRSKRSRLLH SDYMNMTPRR PGPTRKHYQPYAPPRDFAAY RSKKVAKKPT NKAPHPKQEP QEINFPDDLP GSNTAAPVQETLHGCQPVTQ EDGKESRISV QERQ 289 CD8a_P4_sMALPVTALLL PLALLLHAAR PQVQLQQSGP GLVTPSQTLS LTCAISGDSV pCD8SSNSATWNWI RQSPSRGLEW LGRTYYRSKW YNDYAVSVKS RMSINPDTSK _CD28_NQFSLQLNSV TPEDTAVYYC ARGMMTYYYG MDVWGQGTTV TVSSGGGGSG CD40GGGSGGGGSQ PVLTQSSSLS ASPGASASLT CTLRSGINVG PYRIYWYQQKPGSPPQYLLN YKSDSDKQQG SGVPSRFSGS KDASANAGVL LISGLRSEDEADYYCMIWHS SAAVFGGGTQ LTVLSAAAGS GGSGFVPVFL PAKPTTTPAPRPPTPAPTIA SQPLSLRPEA CRPAAGGAVH TRGLDFACDI YIWAPLAGTCGVLLLSLVIT LYCNHRNRSK RSRLLHSDYM NMTPRRPGPT RKHYQPYAPPRDFAAYRSKK VAKKPTNKAP HPKQEPQEIN FPDDLPGSNT AAPVQETLHGCQPVTQEDGK ESRISVQERQ 290 CD2_P4_spMSFPCKFVAS FLLIFNVSSK GAVSQVQLQQ SGPGLVTPSQ TLSLTCAISG CD8DSVSSNSATW NWIRQSPSRG LEWLGRTYYR SKWYNDYAVS VKSRMSINPD _CD28_TSKNQFSLQL NSVTPEDTAV YYCARGMMTY YYGMDVWGQG TTVTVSSGGG CD40GSGGGGSGGG GSQPVLTQSS SLSASPGASA SLTCTLRSGI NVGPYRIYWYQQKPGSPPQY LLNYKSDSDK QQGSGVPSRF SGSKDASANA GVLLISGLRSEDEADYYCMI WHSSAAVFGG GTQLTVLSAA AGSGGSGFVP VFLPAKPTTTPAPRPPTPAP TIASQPLSLR PEACRPAAGG AVHTRGLDFA CDIYIWAPLAGTCGVLLLSL VITLYCNHRN RSKRSRLLHS DYMNMTPRRP GPTRKHYQPYAPPRDFAAYR SKKVAKKPTN KAPHPKQEPQ EINFPDDLPG SNTAAPVQETLHGCQPVTQE DGKESRISVQ ERQ 291 IL2_P4_spMYRMQLLSCI ALSLALVTNS QVQLQQSGPG LVTPSQTLSL TCAISGDSVS CD8SNSATWNWIR QSPSRGLEWL GRTYYRSKWY NDYAVSVKSR MSINPDTSKN _CD28_QFSLQLNSVT PEDTAVYYCA RGMMTYYYGM DVWGQGTTVT VSSGGGGSGG CD40GGSGGGGSQP VLTQSSSLSA SPGASASLTC TLRSGINVGP YRIYWYQQKPGSPPQYLLNY KSDSDKQQGS GVPSRFSGSK DASANAGVLL ISGLRSEDEADYYCMIWHSS AAVFGGGTQL TVLSAAAGSG GSGFVPVFLP AKPTTTPAPRPPTPAPTIAS QPLSLRPEAC RPAAGGAVHT RGLDFACDIY IWAPLAGTCGVLLLSLVITL YCNHRNRSKR SRLLHSDYMN MTPRRPGPTR KHYQPYAPPRDFAAYRSKKV AKKPTNKAPH PKQEPQEINF PDDLPGSNTA APVQETLHGCQPVTQEDGKE SRISVQERQ 292 GM-MWLQSLLLLG TVACSISQVQ LQQSGPGLVT PSQTLSLTCA ISGDSVSSNS CSF_P4_spATWNWIRQSP SRGLEWLGRT YYRSKWYNDY AVSVKSRMSI NPDTSKNQFS CD8LQLNSVTPED TAVYYCARGM MTYYYGMDVW GQGTTVTVSS GGGGSGGGGS _CD28_GGGGSQPVLT QSSSLSASPG ASASLTCTLR SGINVGPYRI YWYQQKPGSP CD40PQYLLNYKSD SDKQQGSGVP SRFSGSKDAS ANAGVLLISG LRSEDEADYYCMIWHSSAAV FGGGTQLTVL SAAAGSGGSG FVPVFLPAKP TTTPAPRPPTPAPTIASQPL SLRPEACRPA AGGAVHTRGL DFACDIYIWA PLAGTCGVLLLSLVITLYCN HRNRSKRSRL LHSDYMNMTP RRPGPTRKHY QPYAPPRDFAAYRSKKVAKK PTNKAPHPKQ EPQEINFPDD LPGSNTAAPV QETLHGCQPV TQEDGKESRI SVQERQ293 hIgGk- MEAPAQLLFL LLLWLPDTTR QVQLQQSGPG LVTPSQTLSL TCAISGDSVS VIIISNSATWNWIR QSPSRGLEWL GRTYYRSKWY NDYAVSVKSR MSINPDTSKN _P4_spCD8QFSLQLNSVT PEDTAVYYCA RGMMTYYYGM DVWGQGTTVT VSSGGGGSGG _CD28_GGSGGGGSQP VLTQSSSLSA SPGASASLTC TLRSGINVGP YRIYWYQQKP CD40GSPPQYLLNY KSDSDKQQGS GVPSRFSGSK DASANAGVLL ISGLRSEDEADYYCMIWHSS AAVFGGGTQL TVLSAAAGSG GSGFVPVFLP AKPTTTPAPRPPTPAPTIAS QPLSLRPEAC RPAAGGAVHT RGLDFACDIY IWAPLAGTCGVLLLSLVITL YCNHRNRSKR SRLLHSDYMN MTPRRPGPTR KHYQPYAPPRDFAAYRSKKV AKKPTNKAPH PKQEPQEINF PDDLPGSNTA APVQETLHGCQPVTQEDGKE SRISVQERQ 294 OSM_P4_CDMGVLLTQRTL LSLVLALLFP SMASMQVOLQ QSGPGLVTPS QTLSLTCAIS 28TMGDSVSSNSAT WNWIRQSPSR GLEWLGRTYY RSKWYNDYAV SVKSRMSINP _CD28_DTSKNQFSLQ LNSVTPEDTA VYYCARGMMT YYYGMDVWGQ GTTVTVSSGG CD40GGSGGGGSGG GGSQPVLTQS SSLSASPGAS ASLTCTLRSG INVGPYRIYWYQQKPGSPPQ YLLNYKSDSD KQQGSGVPSR FSGSKDASAN AGVLLISGLRSEDEADYYCM IWHSSAAVFG GGTQLTVLSA AAGSGGSGFW VLVVVGGVLACYSLLVTVAF IIFWVRSKRS RLLHSDYMNM TPRRPGPTRK HYQPYAPPRDFAAYRSKKVA KKPTNKAPHP KQEPQEINFP DDLPGSNTAA PVQETLHGCQPVTQEDGKES RISVQERQ 295 CD8a_P4_CMALPVTALLL PLALLLHAAR PQVQLQQSGP GLVTPSQTLS LTCAISGDSV D28TMSSNSATWNWI RQSPSRGLEW LGRTYYRSKW YNDYAVSVKS RMSINPDTSK _CD28_NQFSLQLNSV TPEDTAVYYC ARGMMTYYYG MDVWGQGTTV TVSSGGGGSG CD40GGGSGGGGSQ PVLTQSSSLS ASPGASASLT CTLRSGINVG PYRIYWYQQKPGSPPQYLLN YKSDSDKQQG SGVPSRFSGS KDASANAGVL LISGLRSEDEADYYCMIWHS SAAVFGGGTQ LTVLSAAAGS GGSGFWVLVV VGGVLACYSLLVTVAFIIFW VRSKRSRLLH SDYMNMTPRR PGPTRKHYQP YAPPRDFAAYRSKKVAKKPT NKAPHPKQEP QEINFPDDLP GSNTAAPVQE TLHGCQPVTQ EDGKESRISV QERQ296 CD2_P4_CD MSFPCKFVAS FLLIFNVSSK GAVSQVQLQQ SGPGLVTPSQ TLSLTCAISG28TM DSVSSNSATW NWIRQSPSRG LEWLGRTYYR SKWYNDYAVS VKSRMSINPD _CD28_TSKNQFSLQL NSVTPEDTAV YYCARGMMTY YYGMDVWGQG TTVTVSSGGG CD40GSGGGGSGGG GSQPVLTQSS SLSASPGASA SLTCTLRSGI NVGPYRIYWYQQKPGSPPQY LLNYKSDSDK QQGSGVPSRF SGSKDASANA GVLLISGLRSEDEADYYCMI WHSSAAVFGG GTQLTVLSAA AGSGGSGFWV LVVVGGVLACYSLLVTVAFI IFWVRSKRSR LLHSDYMNMT PRRPGPTRKH YQPYAPPRDFAAYRSKKVAK KPTNKAPHPK QEPQEINFPD DLPGSNTAAP VQETLHGCQPVTQEDGKESR ISVQERQ 297 IL2_P4_CDMYRMQLLSCI ALSLALVTNS QVQLQQSGPG LVTPSQTLSL TCAISGDSVS 28TMSNSATWNWIR QSPSRGLEWL GRTYYRSKWY NDYAVSVKSR MSINPDTSKN _CD28_QFSLQLNSVT PEDTAVYYCA RGMMTYYYGM DVWGQGTTVT VSSGGGGSGG CD40GGSGGGGSQP VLTQSSSLSA SPGASASLTC TLRSGINVGP YRIYWYQQKPGSPPQYLLNY KSDSDKQQGS GVPSRFSGSK DASANAGVLL ISGLRSEDEADYYCMIWHSS AAVFGGGTQL TVLSAAAGSG GSGFWVLVVV GGVLACYSLLVTVAFIIFWV RSKRSRLLHS DYMNMTPRRP GPTRKHYQPY APPRDFAAYRSKKVAKKPTN KAPHPKQEPQ EINFPDDLPG SNTAAPVQET LHGCQPVTQE DGKESRISVQ ERQ298 GM-CSF_P4 MWLQSLLLLG TVACSISQVQ LQQSGPGLVT PSQTLSLTCA ISGDSVSSNS_CD28TM ATWNWIRQSP SRGLEWLGRT YYRSKWYNDY AVSVKSRMSI NPDTSKNQFS _CD28_LQLNSVTPED TAVYYCARGM MTYYYGMDVW GQGTTVTVSS GGGGSGGGGS CD40GGGGSQPVLT QSSSLSASPG ASASLTCTLR SGINVGPYRI YWYQQKPGSPPQYLLNYKSD SDKQQGSGVP SRFSGSKDAS ANAGVLLISG LRSEDEADYYCMIWHSSAAV FGGGTQLTVL SAAAGSGGSG FWVLVVVGGV LACYSLLVTVAFIIFWVRSK RSRLLHSDYM NMTPRRPGPT RKHYQPYAPP RDFAAYRSKKVAKKPTNKAP HPKQEPQEIN FPDDLPGSNT AAPVQETLHG CQPVTQEDGK ESRISVQERQ 299hIgGk- MEAPAQLLFL LLLWLPDTTR QVQLQQSGPG LVTPSQTLSL TCAISGDSVS VIIISNSATWNWIR QSPSRGLEWL GRTYYRSKWY NDYAVSVKSR MSINPDTSKN _P4_QFSLQLNSVT PEDTAVYYCA RGMMTYYYGM DVWGQGTTVT VSSGGGGSGG CD28TMGGSGGGGSQP VLTQSSSLSA SPGASASLTC TLRSGINVGP YRIYWYQQKP _CD28_GSPPQYLLNY KSDSDKQQGS GVPSRFSGSK DASANAGVLL ISGLRSEDEA CD40DYYCMIWHSS AAVFGGGTQL TVLSAAAGSG GSGFWVLVVV GGVLACYSLLVTVAFIIFWV RSKRSRLLHS DYMNMTPRRP GPTRKHYQPY APPRDFAAYRSKKVAKKPTN KAPHPKQEPQ EINFPDDLPG SNTAAPVQET LHGCQPVTQE DGKESRISVQ ERQ300 OSMSS MGVLLTQRTL LSLVLALLFP SMASMQVOLQ QSGPELEKPG ASVKLSCKAS_spCD28(t GYSFTGYTMN WVKQSHGKSL EWIGLITPYN GASSYNQKFR GKATLTVDKS run)SSTAYMDLLS LTSEDSAVYF CARGGYDGRG FDYWGQGTTV TVSSGGGGSG _CD28_GGGSGGGGSD IELTQSPAIM SASPGEKVTM TCSASSSVSY MHWYQQKSGT CD40SPKRWIYDTS KLASGVPGRF SGSGSGNSYS LTISSVEAED DATYYCQQWS CTP248KHPLTFGAGT KLEIKAAAGS GGSGIIHVKG KHLCPSPLFP GPSKPFWVLVVVGGVLACYS LLVTVAFIIF WVRSKRSRLL HSDYMNMTPR RPGPTRKHYQPYAPPRDFAA YRSKKVAKKP TNKAPHPKQE PQEINFPDDL PGSNTAAPVQETLHGCQPVT QEDGKESRIS VQERQ 301 CD8a_SS1MALPVTALLL PLALLLHAAR PQVQLQQSGP ELEKPGASVK LSCKASGYSF _spCD28(tTGYTMNWVKQ SHGKSLEWIG LITPYNGASS YNQKFRGKAT LTVDKSSSTA run)YMDLLSLTSE DSAVYFCARG GYDGRGFDYW GQGTTVTVSS GGGGSGGGGS _CD28_CD4GGGGSDIELT QSPAIMSASP GEKVTMTCSA SSSVSYMHWY QQKSGTSPKR 0WIYDTSKLAS GVPGRFSGSG SGNSYSLTIS SVEAEDDATY YCQQWSKHPLTFGAGTKLEI KAAAGSGGSG IIHVKGKHLC PSPLFPGPSK PFWVLVVVGGVLACYSLLVT VAFIIFWVRS KRSRLLHSDY MNMTPRRPGP TRKHYQPYAPPRDFAAYRSK KVAKKPTNKA PHPKQEPQEI NFPDDLPGSN TAAPVQETLHGCQPVTQEDG KESRISVQER Q 302 CD2_SS1MSFPCKFVAS FLLIFNVSSK GAVSQVQLQQ SGPELEKPGA SVKLSCKASG _spCD28(tYSFTGYTMNW VKQSHGKSLE WIGLITPYNG ASSYNQKFRG KATLTVDKSS run)STAYMDLLSL TSEDSAVYFC ARGGYDGRGF DYWGQGTTVT VSSGGGGSGG _CD28_GGSGGGGSDI ELTQSPAIMS ASPGEKVTMT CSASSSVSYM HWYQQKSGTS CD40PKRWIYDTSK LASGVPGRFS GSGSGNSYSL TISSVEAEDD ATYYCQQWSKHPLTFGAGTK LEIKAAAGSG GSGIIHVKGK HLCPSPLFPG PSKPFWVLVVVGGVLACYSL LVTVAFIIFW VRSKRSRLLH SDYMNMTPRR PGPTRKHYQPYAPPRDFAAY RSKKVAKKPT NKAPHPKQEP QEINFPDDLP GSNTAAPVQETLHGCQPVTQ EDGKESRISV QERQ 303 IL2_SS1MYRMQLLSCI ALSLALVTNS QVQLQQSGPE LEKPGASVKL SCKASGYSFT _spCD28(tGYTMNWVKQS HGKSLEWIGL ITPYNGASSY NQKFRGKATL TVDKSSSTAY run)MDLLSLTSED SAVYFCARGG YDGRGFDYWG QGTTVTVSSG GGGSGGGGSG _CD28_GGGSDIELTQ SPAIMSASPG EKVTMTCSAS SSVSYMHWYQ QKSGTSPKRW CD40IYDTSKLASG VPGRFSGSGS GNSYSLTISS VEAEDDATYY CQQWSKHPLTFGAGTKLEIK AAAGSGGSGI IHVKGKHLCP SPLFPGPSKP FWVLVVVGGVLACYSLLVTV AFIIFWVRSK RSRLLHSDYM NMTPRRPGPT RKHYQPYAPPRDFAAYRSKK VAKKPTNKAP HPKQEPQEIN FPDDLPGSNT AAPVQETLHGCQPVTQEDGK ESRISVQERQ 304 GM-MWLQSLLLLG TVACSISQVQ LQQSGPELEK PGASVKLSCK ASGYSFTGYT CSF_SS1MNWVKQSHGK SLEWIGLITP YNGASSYNQK FRGKATLTVD KSSSTAYMDL _spCD28(tLSLTSEDSAV YFCARGGYDG RGFDYWGQGT TVTVSSGGGG SGGGGSGGGG run)SDIELTQSPA IMSASPGEKV TMTCSASSSV SYMHWYQQKS GTSPKRWIYD _CD28_TSKLASGVPG RFSGSGSGNS YSLTISSVEA EDDATYYCQQ WSKHPLTFGA CD40GTKLEIKAAA GSGGSGIIHV KGKHLCPSPL FPGPSKPFWV LVVVGGVLACYSLLVTVAFI IFWVRSKRSR LLHSDYMNMT PRRPGPTRKH YQPYAPPRDFAAYRSKKVAK KPTNKAPHPK QEPQEINFPD DLPGSNTAAP VQETLHGCQPVTQEDGKESR ISVQERQ 305 hIgGk-MEAPAQLLFL LLLWLPDTTR QVQLQQSGPE LEKPGASVKL SCKASGYSFT VIIIGYTMNWVKQS HGKSLEWIGL ITPYNGASSY NQKFRGKATL TVDKSSSTAY _SS1MDLLSLTSED SAVYFCARGG YDGRGFDYWG QGTTVTVSSG GGGSGGGGSG _spCD28(tGGGSDIELTQ SPAIMSASPG EKVTMTCSAS SSVSYMHWYQ QKSGTSPKRW run)IYDTSKLASG VPGRFSGSGS GNSYSLTISS VEAEDDATYY CQQWSKHPLT _CD28_FGAGTKLEIK AAAGSGGSGI IHVKGKHLCP SPLFPGPSKP FWVLVVVGGV CD40LACYSLLVTV AFIIFWVRSK RSRLLHSDYM NMTPRRPGPT RKHYQPYAPPRDFAAYRSKK VAKKPTNKAP HPKQEPQEIN FPDDLPGSNT AAPVQETLHGCQPVTQEDGK ESRISVQERQ 306 OSM_M5MGVLLTQRTL LSLVLALLFP SMASMQVQLV QSGAEVEKPG ASVKVSCKAS _spCD28(tGYTFTDYYMH WVRQAPGQGL EWMGWINPNS GGTNYAQKFQ GRVTMTRDTS run)ISTAYMELSR LRSDDTAVYY CASGWDFDYW GQGTLVTVSS GGGGSGGGGS _CD28_CD4GGGGSDIVMT QSPSSLSASV GDRVTITCRA SQSIRYYLSW YQQKPGKAPK 0LLIYTASILQ NGVPSRFSGS GSGTDFTLTI SSLQPEDFAT YYCLQTYTTP CTP249DFGPGTKVEI KAAAGSGGSG IIHVKGKHLC PSPLFPGPSK PFWVLVVVGGVLACYSLLVT VAFIIFWVRS KRSRLLHSDY MNMTPRRPGP TRKHYQPYAPPRDFAAYRSK KVAKKPTNKA PHPKQEPQEI NFPDDLPGSN TAAPVQETLHGCQPVTQEDG KESRISVQER Q 307 CD8a_M5MALPVTALLL PLALLLHAAR PQVQLVQSGA EVEKPGASVK VSCKASGYTF _spCD28(tTDYYMHWVRQ APGQGLEWMG WINPNSGGTN YAQKFQGRVT MTRDTSISTA run)YMELSRLRSD DTAVYYCASG WDFDYWGQGT LVTVSSGGGG SGGGGSGGGG _CD28_CD4SDIVMTQSPS SLSASVGDRV TITCRASQSI RYYLSWYQQK PGKAPKLLIY 0TASILQNGVP SRFSGSGSGT DFTLTISSLQ PEDFATYYCL QTYTTPDFGPGTKVEIKAAA GSGGSGIIHV KGKHLCPSPL FPGPSKPFWV LVVVGGVLACYSLLVTVAFI IFWVRSKRSR LLHSDYMNMT PRRPGPTRKH YQPYAPPRDFAAYRSKKVAK KPTNKAPHPK QEPQEINFPD DLPGSNTAAP VQETLHGCQPVTQEDGKESR ISVQERQ 308 CD2_M5MSFPCKFVAS FLLIFNVSSK GAVSQVQLVQ SGAEVEKPGA SVKVSCKASG _spCD28(tYTFTDYYMHW VRQAPGQGLE WMGWINPNSG GTNYAQKFQG RVTMTRDTSI run)STAYMELSRL RSDDTAVYYC ASGWDFDYWG QGTLVTVSSG GGGSGGGGSG _CD28_CD4GGGSDIVMTQ SPSSLSASVG DRVTITCRAS QSIRYYLSWY QQKPGKAPKL 0LIYTASILQN GVPSRFSGSG SGTDFTLTIS SLQPEDFATY YCLQTYTTPDFGPGTKVEIK AAAGSGGSGI IHVKGKHLCP SPLFPGPSKP FWVLVVVGGVLACYSLLVTV AFIIFWVRSK RSRLLHSDYM NMTPRRPGPT RKHYQPYAPPRDFAAYRSKK VAKKPTNKAP HPKQEPQEIN FPDDLPGSNT AAPVQETLHGCQPVTQEDGK ESRISVQERQ 309 IL2_M5MYRMQLLSCI ALSLALVTNS QVQLVQSGAE VEKPGASVKV SCKASGYTFT _spCD28(tDYYMHWVRQA PGQGLEWMGW INPNSGGTNY AQKFQGRVTM TRDTSISTAY run)MELSRLRSDD TAVYYCASGW DFDYWGQGTL VTVSSGGGGS GGGGSGGGGS _CD28_DIVMTQSPSS LSASVGDRVT ITCRASQSIR YYLSWYQQKP GKAPKLLIYT CD40ASILQNGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCLQ TYTTPDFGPGTKVEIKAAAG SGGSGIIHVK GKHLCPSPLF PGPSKPFWVL VVVGGVLACYSLLVTVAFII FWVRSKRSRL LHSDYMNMTP RRPGPTRKHY QPYAPPRDFAAYRSKKVAKK PTNKAPHPKQ EPQEINFPDD LPGSNTAAPV QETLHGCQPV TQEDGKESRI SVQERQ310 GM-CSF_M5 MWLQSLLLLG TVACSISQVQ LVQSGAEVEK PGASVKVSCK ASGYTFTDYY_spCD28(t MHWVRQAPGQ GLEWMGWINP NSGGTNYAQK FQGRVTMTRD TSISTAYMEL run)SRLRSDDTAV YYCASGWDFD YWGQGTLVTV SSGGGGSGGG GSGGGGSDIV _CD28_MTQSPSSLSA SVGDRVTITC RASQSIRYYL SWYQQKPGKA PKLLIYTASI CD40LQNGVPSRFS GSGSGTDFTL TISSLQPEDF ATYYCLQTYT TPDFGPGTKVEIKAAAGSGG SGIIHVKGKH LCPSPLFPGP SKPFWVLVVV GGVLACYSLLVTVAFIIFWV RSKRSRLLHS DYMNMTPRRP GPTRKHYQPY APPRDFAAYRSKKVAKKPTN KAPHPKQEPQ EINFPDDLPG SNTAAPVQET LHGCQPVTQE DGKESRISVQ ERQ311 hIgGk- MEAPAQLLFL LLLWLPDTTR QVQLVQSGAE VEKPGASVKV SCKASGYTFT VIIIDYYMHWVRQA PGQGLEWMGW INPNSGGTNY AQKFQGRVTM TRDTSISTAY _M5MELSRLRSDD TAVYYCASGW DFDYWGQGTL VTVSSGGGGS GGGGSGGGGS _spCD28(tDIVMTQSPSS LSASVGDRVT ITCRASQSIR YYLSWYQQKP GKAPKLLIYT run)ASILQNGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCLQ TYTTPDFGPG _CD28_TKVEIKAAAG SGGSGIIHVK GKHLCPSPLF PGPSKPFWVL VVVGGVLACY CD40SLLVTVAFII FWVRSKRSRL LHSDYMNMTP RRPGPTRKHY QPYAPPRDFAAYRSKKVAKK PTNKAPHPKQ EPQEINFPDD LPGSNTAAPV QETLHGCQPV TQEDGKESRI SVQERQ312 OSM_HN1 MGVLLTQRTL LSLVLALLFP SMASMQVQLV QSGAEVKRPG ASVQVSCRAS_spCD28(t GYSINTYYMQ WVRQAPGAGL EWMGVINPSG VTSYAQKFQG RVTLTNDTST run)NTVYMQLNSL TSADTAVYYC ARWALWGDFG MDVWGKGTLV TVSSGGGGSG _CD28_GGGSGGGGSD IQMTQSPSTL SASIGDRVTI TGRASEGIYH WLAWYQQKPG CD40KAPKLLIYKA SSLASGAPSR FSGSGSGTDF TLTISSLQPD DFATYYCQQY CTP250SNYPLTFGGG TKLEIKAAAG SGGSGIIHVK GKHLCPSPLF PGPSKPFWVLVVVGGVLACY SLLVTVAFII FWVRSKRSRL LHSDYMNMTP RRPGPTRKHYQPYAPPRDFA AYRSKKVAKK PTNKAPHPKQ EPQEINFPDD LPGSNTAAPVQETLHGCQPV TQEDGKESRI SVQERQ 313 CD8a_HNlMALPVTALLL PLALLLHAAR PQVQLVQSGA EVKRPGASVQ VSCRASGYSI _spCD28(tNTYYMQWVRQ APGAGLEWMG VINPSGVTSY AQKFQGRVTL TNDTSTNTVY run)MQLNSLTSAD TAVYYCARWA LWGDFGMDVW GKGTLVTVSS GGGGSGGGGS _CD28_GGGGSDIQMT QSPSTLSASI GDRVTITCRA SEGIYHWLAW YQQKPGKAPK CD40LLIYKASSLA SGAPSRFSGS GSGTDFTLTI SSLQPDDFAT YYCQQYSNYPLTFGGGTKLE IKAAAGSGGS GIIHVKGKHL CPSPLFPGPS KPFWVLVVVGGVLACYSLLV TVAFIIFWVR SKRSRLLHSD YMNMTPRRPG PTRKHYQPYAPPRDFAAYRS KKVAKKPTNK APHPKQEPQE INFPDDLPGS NTAAPVQETLHGCQPVTQED GKESRISVQE RQ 314 CD2_HN1MSFPCKFVAS FLLIFNVSSK GAVSQVQLVQ SGAEVKRPGA SVQVSCRASG _spCD28(tYSINTYYMQW VRQAPGAGLE WMGVINPSGV TSYAQKFQGR VTLTNDTSTN run)TVYMQLNSLT SADTAVYYCA RWALWGDFGM DVWGKGTLVT VSSGGGGSGG _CD28_GGSGGGGSDI QMTQSPSTLS ASIGDRVTIT CRASEGIYHW LAWYQQKPGK CD40APKLLIYKAS SLASGAPSRF SGSGSGTDFT LTISSLQPDD FATYYCQQYSNYPLTFGGGT KLEIKAAAGS GGSGIIHVKG KHLCPSPLFP GPSKPFWVLVVVGGVLACYS LLVTVAFIIF WVRSKRSRLL HSDYMNMTPR RPGPTRKHYQPYAPPRDFAA YRSKKVAKKP TNKAPHPKQE PQEINFPDDL PGSNTAAPVQETLHGCQPVT QEDGKESRIS VQERQ 315 IL2_HN1MYRMQLLSCI ALSLALVTNS QVQLVQSGAE VKRPGASVQV SCRASGYSIN _spCD28(tTYYMQWVRQA PGAGLEWMGV INPSGVTSYA QKFQGRVTLT NDTSTNTVYM run)QLNSLTSADT AVYYCARWAL WGDFGMDVWG KGTLVTVSSG GGGSGGGGSG _CD28_GGGSDIQMTQ SPSTLSASIG DRVTITCRAS EGIYHWLAWY QQKPGKAPKL CD40LIYKASSLAS GAPSRFSGSG SGTDFTLTIS SLQPDDFATY YCQQYSNYPLTFGGGTKLEI KAAAGSGGSG IIHVKGKHLC PSPLFPGPSK PFWVLVVVGGVLACYSLLVT VAFIIFWVRS KRSRLLHSDY MNMTPRRPGP TRKHYQPYAPPRDFAAYRSK KVAKKPTNKA PHPKQEPQEI NFPDDLPGSN TAAPVQETLHGCQPVTQEDG KESRISVQER Q 316 GM-MWLQSLLLLG TVACSISQVQ LVQSGAEVKR PGASVQVSCR ASGYSINTYY CSF_HN1MQWVRQAPGA GLEWMGVINP SGVTSYAQKF QGRVTLTNDT STNTVYMQLN _spCD28(tSLTSADTAVY YCARWALWGD FGMDVWGKGT LVTVSSGGGG SGGGGSGGGG run)SDIQMTQSPS TLSASIGDRV TITCRASEGI YHWLAWYQQK PGKAPKLLIY _CD28_KASSLASGAP SRFSGSGSGT DFTLTISSLQ PDDFATYYCQ QYSNYPLTFG CD40GGTKLEIKAA AGSGGSGIIH VKGKHLCPSP LFPGPSKPFW VLVVVGGVLACYSLLVTVAF IIFWVRSKRS RLLHSDYMNM TPRRPGPTRK HYQPYAPPRDFAAYRSKKVA KKPTNKAPHP KQEPQEINFP DDLPGSNTAA PVQETLHGCQPVTQEDGKES RISVQERQ 317 hIgGk-MEAPAQLLFL LLLWLPDTTR QVQLVQSGAE VKRPGASVQV SCRASGYSIN VIIITYYMQWVRQA PGAGLEWMGV INPSGVTSYA QKFQGRVTLT NDTSTNTVYM HNQLNSLTSADT AVYYCARWAL WGDFGMDVWG KGTLVTVSSG GGGSGGGGSG _spCD28(tGGGSDIQMTQ SPSTLSASIG DRVTITCRAS EGIYHWLAWY QQKPGKAPKL run)LIYKASSLAS GAPSRFSGSG SGTDFTLTIS SLQPDDFATY YCQQYSNYPL _CD28_TFGGGTKLEI KAAAGSGGSG IIHVKGKHLC PSPLFPGPSK PFWVLVVVGG CD40VLACYSLLVT VAFIIFWVRS KRSRLLHSDY MNMTPRRPGP TRKHYQPYAPPRDFAAYRSK KVAKKPTNKA PHPKQEPQEI NFPDDLPGSN TAAPVQETLHGCQPVTQEDG KESRISVQER Q 318 OSM_M912MGVLLTQRTL LSLVLALLFP SMASMQVOLQ ESGPGLVKPS ETLSLTCTVS _spCD28(tGGSVSSGSYY WSWIRQPPGK GLEWIGYIYY SGSTNYNPSL KSRVTISVDT run)SKNQFSLKLS SVTAADTAVY YCAREGKNGA FDIWGQGTMV TVSSGGGGSG _CD28_GGGSGGGGSD IQMTQSPSSL SASVGDRVTI TCRASQSISS YLNWYQQKPG CD40KAPKLLIYAA SSLQSGVPSG FSGSGSGTDF TLTISSLQPE DFATYYCQQS CTP251YSTPLTFGGG TKVEIKAAAG SGGSGIIHVK GKHLCPSPLF PGPSKPFWVLVVVGGVLACY SLLVTVAFII FWVRSKRSRL LHSDYMNMTP RRPGPTRKHYQPYAPPRDFA AYRSKKVAKK PTNKAPHPKQ EPQEINFPDD LPGSNTAAPVQETLHGCQPV TQEDGKESRI SVQERQ 319 CD8a_M912MALPVTALLL PLALLLHAAR PQVQLQESGP GLVKPSETLS LTCTVSGGSV _spCD28(tSSGSYYWSWI RQPPGKGLEW IGYIYYSGST NYNPSLKSRV TISVDTSKNQ run)FSLKLSSVTA ADTAVYYCAR EGKNGAFDIW GQGTMVTVSS GGGGSGGGGS _CD28_GGGGSDIQMT QSPSSLSASV GDRVTITCRA SQSISSYLNW YQQKPGKAPK CD40LLIYAASSLQ SGVPSGFSGS GSGTDFTLTI SSLQPEDFAT YYCQQSYSTPLTFGGGTKVE IKAAAGSGGS GIIHVKGKHL CPSPLFPGPS KPFWVLVVVGGVLACYSLLV TVAFIIFWVR SKRSRLLHSD YMNMTPRRPG PTRKHYQPYAPPRDFAAYRS KKVAKKPTNK APHPKQEPQE INFPDDLPGS NTAAPVQETLHGCQPVTQED GKESRISVQE RQ 320 CD2_M912MSFPCKFVAS FLLIFNVSSK GAVSQVQLQE SGPGLVKPSE TLSLTCTVSG _spCD28(tGSVSSGSYYW SWIRQPPGKG LEWIGYIYYS GSTNYNPSLK SRVTISVDTS run)KNQFSLKLSS VTAADTAVYY CAREGKNGAF DIWGQGTMVT VSSGGGGSGG _CD28_GGSGGGGSDI QMTQSPSSLS ASVGDRVTIT CRASQSISSY LNWYQQKPGK CD40APKLLIYAAS SLQSGVPSGF SGSGSGTDFT LTISSLQPED FATYYCQQSYSTPLTFGGGT KVEIKAAAGS GGSGIIHVKG KHLCPSPLFP GPSKPFWVLVVVGGVLACYS LLVTVAFIIF WVRSKRSRLL HSDYMNMTPR RPGPTRKHYQPYAPPRDFAA YRSKKVAKKP TNKAPHPKQE PQEINFPDDL PGSNTAAPVQETLHGCQPVT QEDGKESRIS VQERQ 321 IL2_M912MYRMQLLSCI ALSLALVTNS QVQLQESGPG LVKPSETLSL TCTVSGGSVS _spCD28(tSGSYYWSWIR QPPGKGLEWI GYIYYSGSTN YNPSLKSRVT ISVDTSKNQF run)SLKLSSVTAA DTAVYYCARE GKNGAFDIWG QGTMVTVSSG GGGSGGGGSG _CD28_CD4GGGSDIQMTQ SPSSLSASVG DRVTITCRAS QSISSYLNWY QQKPGKAPKL 0LIYAASSLQS GVPSGFSGSG SGTDFTLTIS SLQPEDFATY YCQQSYSTPLTFGGGTKVEI KAAAGSGGSG IIHVKGKHLC PSPLFPGPSK PFWVLVVVGGVLACYSLLVT VAFIIFWVRS KRSRLLHSDY MNMTPRRPGP TRKHYQPYAPPRDFAAYRSK KVAKKPTNKA PHPKQEPQEI NFPDDLPGSN TAAPVQETLHGCQPVTQEDG KESRISVQER Q 322 GM-MWLQSLLLLG TVACSISQVQ LQESGPGLVK PSETLSLTCT VSGGSVSSGS CSF_M912YYWSWIRQPP GKGLEWIGYI YYSGSTNYNP SLKSRVTISV DTSKNQFSLK _spCD28(tLSSVTAADTA VYYCAREGKN GAFDIWGQGT MVTVSSGGGG SGGGGSGGGG run)SDIQMTQSPS SLSASVGDRV TITCRASQSI SSYLNWYQQK PGKAPKLLIY _CD28_AASSLQSGVP SGFSGSGSGT DFTLTISSLQ PEDFATYYCQ QSYSTPLTFG CD40GGTKVEIKAA AGSGGSGIIH VKGKHLCPSP LFPGPSKPFW VLVVVGGVLACYSLLVTVAF IIFWVRSKRS RLLHSDYMNM TPRRPGPTRK HYQPYAPPRDFAAYRSKKVA KKPTNKAPHP KQEPQEINFP DDLPGSNTAA PVQETLHGCQPVTQEDGKES RISVQERQ 323 hIgGk-MEAPAQLLFL LLLWLPDTTR QVQLQESGPG LVKPSETLSL TCTVSGGSVS VIIISGSYYWSWIR QPPGKGLEWI GYIYYSGSTN YNPSLKSRVT ISVDTSKNQF _M912SLKLSSVTAA DTAVYYCARE GKNGAFDIWG QGTMVTVSSG GGGSGGGGSG _spCD28(tGGGSDIQMTQ SPSSLSASVG DRVTITCRAS QSISSYLNWY QQKPGKAPKL run)LIYAASSLQS GVPSGFSGSG SGTDFTLTIS SLQPEDFATY YCQQSYSTPL _CD28_TFGGGTKVEI KAAAGSGGSG IIHVKGKHLC PSPLFPGPSK PFWVLVVVGG CD40VLACYSLLVT VAFIIFWVRS KRSRLLHSDY MNMTPRRPGP TRKHYQPYAPPRDFAAYRSK KVAKKPTNKA PHPKQEPQEI NFPDDLPGSN TAAPVQETLHGCQPVTQEDG KESRISVQER Q 324 OSM_HuYP2MGVLLTQRTL LSLVLALLFP SMASMEVQLV ESGGGLVQPG GSLRLSCAAS 18GFDLGFYFYA CWVRQAPGKG LEWVSCIYTA GSGSTYYASW AKGRFTISRD _spCD28(tNSKNTLYLQM NSLRAEDTAV YYCARSTANT RSTYYLNLWG QGTLVTVSSG run)GGGSGGGGSG GGGSDIQMTQ SPSSLSASVG DRVTITCQAS QRISSYLSWY _CD28_QQKPGKVPKL LIYGASTLAS GVPSRFSGSG SGTDFTLTIS SLQPEDVATY CD40YCQSYAYFDS NNWHAFGGGT KVEIAAAGSG GSGIIHVKGK HLCPSPLFPG CTP252PSKPFWVLVV VGGVLACYSL LVTVAFIIFW VRSKRSRLLH SDYMNMTPRRPGPTRKHYQP YAPPRDFAAY RSKKVAKKPT NKAPHPKQEP QEINFPDDLPGSNTAAPVQE TLHGCQPVTQ EDGKESRISV QERQ 325 CD8a_HuYPMALPVTALLL PLALLLHAAR PEVQLVESGG GLVQPGGSLR LSCAASGFDL 218GFYFYACWVR QAPGKGLEWV SCIYTAGSGS TYYASWAKGR FTISRDNSKN _spCD28(tTLYLQMNSLR AEDTAVYYCA RSTANTRSTY YLNLWGQGTL VTVSSGGGGS run)GGGGSGGGGS DIQMTQSPSS LSASVGDRVT ITCQASQRIS SYLSWYQQKP _CD28_CD4GKVPKLLIYG ASTLASGVPS RFSGSGSGTD FTLTISSLQP EDVATYYCQS 0YAYFDSNNWH AFGGGTKVEI AAAGSGGSGI IHVKGKHLCP SPLFPGPSKPFWVLVVVGGV LACYSLLVTV AFIIFWVRSK RSRLLHSDYM NMTPRRPGPTRKHYQPYAPP RDFAAYRSKK VAKKPTNKAP HPKQEPQEIN FPDDLPGSNTAAPVQETLHG CQPVTQEDGK ESRISVQERQ 326 CD2_HuYP2MSFPCKFVAS FLLIFNVSSK GAVSEVQLVE SGGGLVQPGG SLRLSCAASG 18FDLGFYFYAC WVRQAPGKGL EWVSCIYTAG SGSTYYASWA KGRFTISRDN _spCD28(tSKNTLYLQMN SLRAEDTAVY YCARSTANTR STYYLNLWGQ GTLVTVSSGG run)GGSGGGGSGG GGSDIQMTQS PSSLSASVGD RVTITCQASQ RISSYLSWYQ _CD28_QKPGKVPKLL IYGASTLASG VPSRFSGSGS GTDFTLTISS LQPEDVATYY CD40CQSYAYFDSN NWHAFGGGTK VEIAAAGSGG SGIIHVKGKH LCPSPLFPGPSKPFWVLVVV GGVLACYSLL VTVAFIIFWV RSKRSRLLHS DYMNMTPRRPGPTRKHYQPY APPRDFAAYR SKKVAKKPTN KAPHPKQEPQ EINFPDDLPGSNTAAPVQET LHGCQPVTQE DGKESRISVQ ERQ 327 IL2_HuYP2MYRMQLLSCI ALSLALVTNS EVQLVESGGG LVQPGGSLRL SCAASGFDLG 18FYFYACWVRQ APGKGLEWVS GIYTAGSGST YYASWAKGRF TISRDNSKNT _spCD28(tLYLQMNSLRA EDTAVYYCAR STANTRSTYY LNLWGQGTLV TVSSGGGGSG run)GGGSGGGGSD IQMTQSPSSL SASVGDRVTI TCQASQRISS YLSWYQQKPG _CD28_KVPKLLIYGA STLASGVPSR FSGSGSGTDF TLTISSLQPE DVATYYCQSY CD40AYFDSNNWHA FGGGTKVEIA AAGSGGSGII HVKGKHLCPS PLFPGPSKPFWVLVVVGGVL ACYSLLVTVA FIIFWVRSKR SRLLHSDYMN MTPRRPGPTRKHYQPYAPPR DFAAYRSKKV AKKPTNKAPH PKQEPQEINF PDDLPGSNTAAPVQETLHGC QPVTQEDGKE SRISVQERQ 328 GM-MWLQSLLLLG TVACSISEVQ LVESGGGLVQ PGGSLRLSCA ASGFDLGFYF CSF_HuYP2YACWVRQAPG KGLEWVSCIY TAGSGSTYYA SWAKGRFTIS RDNSKNTLYL 18QMNSLRAEDT AVYYCARSTA NTRSTYYLNL WGQGTLVTVS SGGGGSGGGG _spCD28(tSGGGGSDIQM TQSPSSLSAS VGDRVTITCQ ASQRISSYLS WYQQKPGKVP run)KLLIYGASTL ASGVPSRFSG SGSGTDFTLT ISSLQPEDVA TYYCQSYAYF _CD28_DSNNWHAFGG GTKVEIAAAG SGGSGIIHVK GKHLCPSPLF PGPSKPFWVL CD40VVVGGVLACY SLLVTVAFII FWVRSKRSRL LHSDYMNMTP RRPGPTRKHYQPYAPPRDFA AYRSKKVAKK PTNKAPHPKQ EPQEINFPDD LPGSNTAAPVQETLHGCQPV TQEDGKESRI SVQERQ 329 hIgGk-MEAPAQLLFL LLLWLPDTTR EVQLVESGGG LVQPGGSLRL SCAASGFDLG VIIIFYFYACWVRQ APGKGLEWVS GIYTAGSGST YYASWAKGRF TISRDNSKNT _HuYP218LYLQMNSLRA EDTAVYYCAR STANTRSTYY LNLWGQGTLV TVSSGGGGSG _spCD28(tGGGSGGGGSD IQMTQSPSSL SASVGDRVTI TCQASQRISS YLSWYQQKPG run)KVPKLLIYGA STLASGVPSR FSGSGSGTDF TLTISSLQPE DVATYYCQSY _CD28_AYFDSNNWHA FGGGTKVEIA AAGSGGSGII HVKGKHLCPS PLFPGPSKPF CD40WVLVVVGGVL ACYSLLVTVA FIIFWVRSKR SRLLHSDYMN MTPRRPGPTRKHYQPYAPPR DFAAYRSKKV AKKPTNKAPH PKQEPQEINF PDDLPGSNTAAPVQETLHGC QPVTQEDGKE SRISVQERQ 330 OSM_P4MGVLLTQRTL LSLVLALLFP SMASMQVOLQ QSGPGLVTPS QTLSLTCAIS _spCD28(tGDSVSSNSAT WNWIRQSPSR GLEWLGRTYY RSKWYNDYAV SVKSRMSINP run)DTSKNQFSLQ LNSVTPEDTA VYYCARGMMT YYYGMDVWGQ GTTVTVSSGG _CD28_GGSGGGGSGG GGSQPVLTQS SSLSASPGAS ASLTCTLRSG INVGPYRIYW CD40YQQKPGSPPQ YLLNYKSDSD KQQGSGVPSR FSGSKDASAN AGVLLISGLR CTP253SEDEADYYCM IWHSSAAVFG GGTQLTVLSA AAGSGGSGII HVKGKHLCPSPLFPGPSKPF WVLVVVGGVL ACYSLLVTVA FIIFWVRSKR SRLLHSDYMNMTPRRPGPTR KHYQPYAPPR DFAAYRSKKV AKKPTNKAPH PKQEPQEINFPDDLPGSNTA APVQETLHGC QPVTQEDGKE SRISVQERQ 331 CD8a_P4MALPVTALLL PLALLLHAAR PQVQLQQSGP GLVTPSQTLS LTCAISGDSV _spCD28(tSSNSATWNWI RQSPSRGLEW LGRTYYRSKW YNDYAVSVKS RMSINPDTSK run)NQFSLQLNSV TPEDTAVYYC ARGMMTYYYG MDVWGQGTTV TVSSGGGGSG _CD28_GGGSGGGGSQ PVLTQSSSLS ASPGASASLT CTLRSGINVG PYRIYWYQQK CD40PGSPPQYLLN YKSDSDKQQG SGVPSRFSGS KDASANAGVL LISGLRSEDEADYYCMIWHS SAAVFGGGTQ LTVLSAAAGS GGSGIIHVKG KHLCPSPLFPGPSKPFWVLV VVGGVLACYS LLVTVAFIIF WVRSKRSRLL HSDYMNMTPRRPGPTRKHYQ PYAPPRDFAA YRSKKVAKKP TNKAPHPKQE PQEINFPDDLPGSNTAAPVQ ETLHGCQPVT QEDGKESRIS VQERQ 332 CD2_P4MSFPCKFVAS FLLIFNVSSK GAVSQVQLQQ SGPGLVTPSQ TLSLTCAISG _spCD28(tDSVSSNSATW NWIRQSPSRG LEWLGRTYYR SKWYNDYAVS VKSRMSINPD run)TSKNQFSLQL NSVTPEDTAV YYCARGMMTY YYGMDVWGQG TTVTVSSGGG _CD28_GSGGGGSGGG GSQPVLTQSS SLSASPGASA SLTCTLRSGI NVGPYRIYWY CD40QQKPGSPPQY LLNYKSDSDK QQGSGVPSRF SGSKDASANA GVLLISGLRSEDEADYYCMI WHSSAAVFGG GTQLTVLSAA AGSGGSGIIH VKGKHLCPSPLFPGPSKPFW VLVVVGGVLA CYSLLVTVAF IIFWVRSKRS RLLHSDYMNMTPRRPGPTRK HYQPYAPPRD FAAYRSKKVA KKPTNKAPHP KQEPQEINFPDDLPGSNTAA PVQETLHGCQ PVTQEDGKES RISVQERQ 333 IL2_P4MYRMQLLSCI ALSLALVTNS QVQLQQSGPG LVTPSQTLSL TCAISGDSVS _spCD28(tSNSATWNWIR QSPSRGLEWL GRTYYRSKWY NDYAVSVKSR MSINPDTSKN run)QFSLQLNSVT PEDTAVYYCA RGMMTYYYGM DVWGQGTTVT VSSGGGGSGG _CD28_GGSGGGGSQP VLTQSSSLSA SPGASASLTC TLRSGINVGP YRIYWYQQKP CD40GSPPQYLLNY KSDSDKQQGS GVPSRFSGSK DASANAGVLL ISGLRSEDEADYYCMIWHSS AAVFGGGTQL TVLSAAAGSG GSGIIHVKGK HLCPSPLFPGPSKPFWVLVV VGGVLACYSL LVTVAFIIFW VRSKRSRLLH SDYMNMTPRRPGPTRKHYQP YAPPRDFAAY RSKKVAKKPT NKAPHPKQEP QEINFPDDLPGSNTAAPVQE TLHGCQPVTQ EDGKESRISV QERQ 334 GM-CSF_P4MWLQSLLLLG TVACSISQVQ LQQSGPGLVT PSQTLSLTCA ISGDSVSSNS _spCD28(tATWNWIRQSP SRGLEWLGRT YYRSKWYNDY AVSVKSRMSI NPDTSKNQFS run)LQLNSVTPED TAVYYCARGM MTYYYGMDVW GQGTTVTVSS GGGGSGGGGS _CD28_GGGGSQPVLT QSSSLSASPG ASASLTCTLR SGINVGPYRI YWYQQKPGSP CD40PQYLLNYKSD SDKQQGSGVP SRFSGSKDAS ANAGVLLISG LRSEDEADYYCMIWHSSAAV FGGGTQLTVL SAAAGSGGSG IIHVKGKHLC PSPLFPGPSKPFWVLVVVGG VLACYSLLVT VAFIIFWVRS KRSRLLHSDY MNMTPRRPGPTRKHYQPYAP PRDFAAYRSK KVAKKPTNKA PHPKQEPQEI NFPDDLPGSNTAAPVQETLH GCQPVTQEDG KESRISVQER Q 335 hIgGk-MEAPAQLLFL LLLWLPDTTR QVQLQQSGPG LVTPSQTLSL TCAISGDSVS VIIISNSATWNWIR QSPSRGLEWL GRTYYRSKWY NDYAVSVKSR MSINPDTSKN _P4QFSLQLNSVT PEDTAVYYCA RGMMTYYYGM DVWGQGTTVT VSSGGGGSGG _spCD28(tGGSGGGGSQP VLTQSSSLSA SPGASASLTC TLRSGINVGP YRIYWYQQKP run)GSPPQYLLNY KSDSDKQQGS GVPSRFSGSK DASANAGVLL ISGLRSEDEA _CD28_DYYCMIWHSS AAVFGGGTQL TVLSAAAGSG GSGIIHVKGK HLCPSPLFPG CD40PSKPFWVLVV VGGVLACYSL LVTVAFIIFW VRSKRSRLLH SDYMNMTPRRPGPTRKHYQP YAPPRDFAAY RSKKVAKKPT NKAPHPKQEP QEINFPDDLPGSNTAAPVQE TLHGCQPVTQ EDGKESRISV QERQ 336 NTRK1 ICNKCGRRNKFG INRPAVLAPE DGLAMSLHFM TLGGSSLSPT EGKGSGLQGH domainIIENPQYFSD ACVHHIKRRD IVLKWELGEG AFGKVFLAEC HNLLPEQDKMLVAVKALKEA SESARQDFQR EAELLTMLQH QHIVRFFGVC TEGRPLLMVFEYMRHGDLNR FLRSHGPDAK LLAGGEDVAP GPLGLGQLLA VASQVAAGMVYLAGLHFVHR DLATRNCLVG QGLVVKIGDF GMSRDIYSTD YYRVGGRTMLPIRWMPPESI LYRKFTTESD VWSFGVVLWE IFTYGKQPWY QLSNTEAIDCITQGRELERP RACPPEVYAI MRGCWQREPQ QRHSIKDVHA RLQALAQAPP VYLDVLG 337 ICOSMKSGLWYFFL FLCRIKVLTG EINGSANYEM FIFHNGGVQI LCKYPDIVQQFKMQLLKGGQ ILCDLTKTKG SGNTVSIKSL KFCHSQLSNN SVSFFLYNLDHSHANYYFCN LSIFDPPPFK VTLTGGYLHI YESQLCCQLK FWLPIGCAAFVVVCILGCIL ICWLTKKKYS SSVHDPNGEY MFMRAVNTAK KSRLTDVTL 338 ICOS ICCWLTKKKYSS SVHDPNGEYM FMRAVNTAKK SRLTDVTL domain 339 mouse CD2RDNETIWGVL GHGITLNIPN FQMTDDIDEV RWVRRGTLVA EFKRKKPPFLISETYEVLAN GSLKIKKPMM RNDSGTYNVM VYGTNGMTRL EKDLDVRILERVSKPMIHWE CPNTTLTCAV LQGTDFELKL YQGETLLNSL PQKNMSYQWTNLNAPFKCEA INPVSKESKM EVVNCPEKGL SFYVTVGVGA GGLLLVLLVALFIFCICKRR KRNRRRKDEE LEIKASRTST VERGPKPHST PAAAAQNSVALQAPPPPGHH LQTPGHRPLP PGHRTREHQQ KKRPPPSGTQ IHQQKGPPLPRPRVQPKPPC GSGDGVSLPP PN 340 mouse CD2KRRKRNRRRK DEELEIKASR TSTVERGPKP HSTPAAAAQN SVALQAPPPP IC domainGHHLQTPGHR PLPPGHRTRE HQQKKRPPPS GTQIHQQKGP PLPRPRVQPK PPCGSGDGVS LPPPN341 CD137 IC KRGRKKLLYI FKQPFMRPVQ TTQEEDGCSC RFPEEEEGGC EL domain 342DAP10 IC LCARPRRSPA QEDGKVYINM PGRG domain 343 CD134 ICALYLLRRDQR LPPDAHKPPG GGSFRTPIQE EQADAHSTLA KI domain 344 hMFE23.MGVLLTQRTL LSLVLALLFP SMASMQVKLE QSGAEVVKPG ASVKLSCKAS TMtCD28.NGFNIKDSYMH WLRQGPGQRL EWIGWIDPEN GDTEYAPKFQ GKATFTTDTS TRK1ANTAYLGLSS LRPEDTAVYY CNEGTPTGPY YFDYWGQGTL VTVSSGGGGS CTP313GGGGSGGGGS ENVLTQSPSS MSASVGDRVN IACSASSSVS YMHWFQQKPGKSPKLWIYST SNLASGVPSR FSGSGSGTDY SLTISSMQPE DAATYYCQQRSSYPLTFGGG TKLEIKAAAG SGGSGILVKQ SPMLVAYDNA VNLSCKYSYNLFSREFRASL HKGLDSAVEV CVVYGNYSQQ LQVYSKTGFN CDGKLGNESVTFYLQNLYVN QTDIYFCKIE VMYPPPYLDN EKSNGTIIHV KGKHLCPSPLFPGPSKPFWV LVVVGGVLAC YSLLVTVAFI IFWVNKCGRR NKFGINRPAVLAPEDGLAMS LHFMTLGGSS LSPTEGKGSG LQGHIIENPQ YFSDACVHHIKRRDIVLKWE LGEGAFGKVF LAECHNLLPE QDKMLVAVKA LKEASESARQDFQREAELLT MLQHQHIVRF FGVCTEGRPL LMVFEYMRHG DLNRFLRSHGPDAKLLAGGE DVAPGPLGLG QLLAVASQVA AGMVYLAGLH FVHRDLATRNCLVGQGLVVK IGDFGMSRDI YSTDYYRVGG RTMLPIRWMP PESILYRKFTTESDVWSFGV VLWEIFTYGK QPWYQLSNTE AIDCITQGRE LERPRACPPEVYAIMRGCWQ REPQQRHSIK DVHARLQALA QAPPVYLDVLG 345 hMFE23.MGVLLTQRTL LSLVLALLFP SMASMQVKLE QSGAEVVKPG ASVKLSCKAS TMtCD28.GFNIKDSYMH WLRQGPGQRL EWIGWIDPEN GDTEYAPKFQ GKATFTTDTS NTRK1.ANTAYLGLSS LRPEDTAVYY CNEGTPTGPY YFDYWGQGTL VTVSSGGGGS CD40GGGGSGGGGS ENVLTQSPSS MSASVGDRVN IACSASSSVS YMHWFQQKPG CTP314KSPKLWIYST SNLASGVPSR FSGSGSGTDY SLTISSMQPE DAATYYCQQRSSYPLTFGGG TKLEIKAAAG SGGSGILVKQ SPMLVAYDNA VNLSCKYSYNLFSREFRASL HKGLDSAVEV CVVYGNYSQQ LQVYSKTGFN CDGKLGNESVTFYLQNLYVN QTDIYFCKIE VMYPPPYLDN EKSNGTIIHV KGKHLCPSPLFPGPSKPFWV LVVVGGVLAC YSLLVTVAFI IFWVNKCGRR NKFGINRPAVLAPEDGLAMS LHFMTLGGSS LSPTEGKGSG LQGHIIENPQ YFSDACVHHIKRRDIVLKWE LGEGAFGKVF LAECHNLLPE QDKMLVAVKA LKEASESARQDFQREAELLT MLQHQHIVRF FGVCTEGRPL LMVFEYMRHG DLNRFLRSHGPDAKLLAGGE DVAPGPLGLG QLLAVASQVA AGMVYLAGLH FVHRDLATRNCLVGQGLVVK IGDFGMSRDI YSTDYYRVGG RTMLPIRWMP PESILYRKFTTESDVWSFGV VLWEIFTYGK QPWYQLSNTE AIDCITQGRE LERPRACPPEVYAIMRGCWQ REPQQRHSIK DVHARLQALA QAPPVYLDVL GKKVAKKPTNKAPHPKQEPQ EINFPDDLPG SNTAAPVQET LHGCQPVTQE DGKESRISVQ ERQ 346 hMFE23.CDMGVLLTQRTL LSLVLALLFP SMASMQVKLE QSGAEVVKPG ASVKLSCKAS 28 .GFNIKDSYMH WLRQGPGQRL EWIGWIDPEN GDTEYAPKFQ GKATFTTDTS tICNTRKIANTAYLGLSS LRPEDTAVYY CNEGTPTGPY YFDYWGQGTL VTVSSGGGGS CTP315GGGGSGGGGS ENVLTQSPSS MSASVGDRVN IACSASSSVS YMHWFQQKPGKSPKLWIYST SNLASGVPSR FSGSGSGTDY SLTISSMQPE DAATYYCQQRSSYPLTFGGG TKLEIKAAAG SGGSGILVKQ SPMLVAYDNA VNLSCKYSYNLFSREFRASL HKGLDSAVEV CVVYGNYSQQ LQVYSKTGFN CDGKLGNESVTFYLQNLYVN QTDIYFCKIE VMYPPPYLDN EKSNGTIIHV KGKHLCPSPLFPGPSKPFWV LVVVGGVLAC YSLLVTVAFI IFWVRSKRSR LLHSDYMNMTPRRPGPTRKH YQPYAPPRDF AAYRSNKCGR RNKFGINRPA VLAPEDGLAMSLHFMTLGGS SLSPTEGKGS GLQGHIIENP QYFSDACVHH IKRRDIVLKWELGEGAFGKV FLAECHNLLP EQDKMLVAVK ALKEASESAR QDFQREAELLTMLQHQHIVR FFGVCTEGRP LLMVFEYMRH GDLNRFLRSH GPDAKLLAGGEDVAPGPLGL GQLLAVASQV AAGMVYLAGL HFVHRDLATR NCLVGQGLVVKIGDFGMSRD IYSTDYYRVG GRTMLPIRWM PPESILYRKF TTESDVWSFGVVLWEIFTYG KQPWYQLSNT EAIDCITQGR ELERPRACPP EVYAIMRGCWQREPQQRHSI KDVHARLQAL AQAPPVYLDV LG 347 hMFE23.CDMGVLLTQRTL LSLVLALLFP SMASMQVKLE QSGAEVVKPG ASVKLSCKAS 28 .GFNIKDSYMH WLRQGPGQRL EWIGWIDPEN GDTEYAPKFQ GKATFTTDTS tICNTRKI.ANTAYLGLSS LRPEDTAVYY CNEGTPTGPY YFDYWGQGTL VTVSSGGGGS CD40GGGGSGGGGS ENVLTQSPSS MSASVGDRVN IACSASSSVS YMHWFQQKPG CTP316KSPKLWIYST SNLASGVPSR FSGSGSGTDY SLTISSMQPE DAATYYCQQRSSYPLTFGGG TKLEIKAAAG SGGSGILVKQ SPMLVAYDNA VNLSCKYSYNLFSREFRASL HKGLDSAVEV CVVYGNYSQQ LQVYSKTGFN CDGKLGNESVTFYLQNLYVN QTDIYFCKIE VMYPPPYLDN EKSNGTIIHV KGKHLCPSPLFPGPSKPFWV LVVVGGVLAC YSLLVTVAFI IFWVRSKRSR LLHSDYMNMTPRRPGPTRKH YQPYAPPRDF AAYRSNKCGR RNKFGINRPA VLAPEDGLAMSLHFMTLGGS SLSPTEGKGS GLQGHIIENP QYFSDACVHH IKRRDIVLKWELGEGAFGKV FLAECHNLLP EQDKMLVAVK ALKEASESAR QDFQREAELLTMLQHQHIVR FFGVCTEGRP LLMVFEYMRH GDLNRFLRSH GPDAKLLAGGEDVAPGPLGL GQLLAVASQV AAGMVYLAGL HFVHRDLATR NCLVGQGLVVKIGDFGMSRD IYSTDYYRVG GRTMLPIRWM PPESILYRKF TTESDVWSFGVVLWEIFTYG KQPWYQLSNT EAIDCITQGR ELERPRACPP EVYAIMRGCWQREPQQRHSI KDVHARLQAL AQAPPVYLDV LGKKVAKKPT NKAPHPKQEPQEINFPDDLP GSNTAAPVQE TLHGCQPVTQ EDGKESRISV QERQ 348 hMFE23.MGVLLTQRTL LSLVLALLFP SMASMQVKLE QSGAEVVKPG ASVKLSCKAS ICOS.CD40GFNIKDSYMH WLRQGPGQRL EWIGWIDPEN GDTEYAPKFQ GKATFTTDTS CTP317ANTAYLGLSS LRPEDTAVYY CNEGTPTGPY YFDYWGQGTL VTVSSGGGGSGGGGSGGGGS ENVLTQSPSS MSASVGDRVN IACSASSSVS YMHWFQQKPGKSPKLWIYST SNLASGVPSR FSGSGSGTDY SLTISSMQPE DAATYYCQQRSSYPLTFGGG TKLEIKAAAG SGGSGGEING SANYEMFIFH NGGVQILCKYPDIVQQFKMQ LLKGGQILCD LTKTKGSGNT VSIKSLKFCH SQLSNNSVSFFLYNLDHSHA NYYFCNLSIF DPPPFKVTLT GGYLHIYESQ LCCQLKFWLPIGCAAFVVVC ILGCILICWL TKKKYSSSVH DPNGEYMFMR AVNTAKKSRLTDVTLKKVAK KPTNKAPHPK QEPQEINFPD DLPGSNTAAP VQETLHGCQPVTQEDGKESR ISVQERQ 349 hMFE23.MGVLLTQRTL LSLVLALLFP SMASMQVKLE QSGAEVVKPG ASVKLSCKAS CD28.ICOSGFNIKDSYMH WLRQGPGQRL EWIGWIDPEN GDTEYAPKFQ GKATFTTDTS CTP318ANTAYLGLSS LRPEDTAVYY CNEGTPTGPY YFDYWGQGTL VTVSSGGGGSGGGGSGGGGS ENVLTQSPSS MSASVGDRVN IACSASSSVS YMHWFQQKPGKSPKLWIYST SNLASGVPSR FSGSGSGTDY SLTISSMQPE DAATYYCQQRSSYPLTFGGG TKLEIKAAAG SGGSGILVKQ SPMLVAYDNA VNLSCKYSYNLFSREFRASL HKGLDSAVEV CVVYGNYSQQ LQVYSKTGFN CDGKLGNESVTFYLQNLYVN QTDIYFCKIE VMYPPPYLDN EKSNGTIIHV KGKHLCPSPLFPGPSKPFWV LVVVGGVLAC YSLLVTVAFI IFWVRSKRSR LLHSDYMNMTPRRPGPTRKH YQPYAPPRDF AAYRSCWLTK KKYSSSVHDP NGEYMFMRAV NTAKKSRLTD VTL350 hMFE23.CD MGVLLTQRTL LSLVLALLFP SMASMQVKLE QSGAEVVKPG ASVKLSCKAS 28.GFNIKDSYMH WLRQGPGQRL EWIGWIDPEN GDTEYAPKFQ GKATFTTDTS ICOS.CD40ANTAYLGLSS LRPEDTAVYY CNEGTPTGPY YFDYWGQGTL VTVSSGGGGS CTP319GGGGSGGGGS ENVLTQSPSS MSASVGDRVN IACSASSSVS YMHWFQQKPGKSPKLWIYST SNLASGVPSR FSGSGSGTDY SLTISSMQPE DAATYYCQQRSSYPLTFGGG TKLEIKAAAG SGGSGILVKQ SPMLVAYDNA VNLSCKYSYNLFSREFRASL HKGLDSAVEV CVVYGNYSQQ LQVYSKTGFN CDGKLGNESVTFYLQNLYVN QTDIYFCKIE VMYPPPYLDN EKSNGTIIHV KGKHLCPSPLFPGPSKPFWV LVVVGGVLAC YSLLVTVAFI IFWVRSKRSR LLHSDYMNMTPRRPGPTRKH YQPYAPPRDF AAYRSCWLTK KKYSSSVHDP NGEYMFMRAVNTAKKSRLTD VTLKKVAKKP TNKAPHPKQE PQEINFPDDL PGSNTAAPVQETLHGCQPVT QEDGKESRIS VQERQ 351 hMFE23.CDMGVLLTQRTL LSLVLALLFP SMASMQVKLE QSGAEVVKPG ASVKLSCKAS 2.CD40GFNIKDSYMH WLRQGPGQRL EWIGWIDPEN GDTEYAPKFQ GKATFTTDTS CTP320ANTAYLGLSS LRPEDTAVYY CNEGTPTGPY YFDYWGQGTL VTVSSGGGGSGGGGSGGGGS ENVLTQSPSS MSASVGDRVN IACSASSSVS YMHWFQQKPGKSPKLWIYST SNLASGVPSR FSGSGSGTDY SLTISSMQPE DAATYYCQQRSSYPLTFGGG TKLEIKAAAG SGGSGRDNET IWGVLGHGIT LNIPNFQMTDDIDEVRWVRR GTLVAEFKRK KPPFLISETY EVLANGSLKI KKPMMRNDSGTYNVMVYGTN GMTRLEKDLD VRILERVSKP MIHWECPNTT LTCAVLQGTDFELKLYQGET LLNSLPQKNM SYQWTNLNAP FKCEAINPVS KESKMEVVNCPEKGLSFYVT VGVGAGGLLL VLLVALFIFC ICKRRKRNRR RKDEELEIKASRTSTVERGP KPHSTPAAAA QNSVALQAPP PPGHHLQTPG HRPLPPGHRTREHQQKKRPP PSGTQIHQQK GPPLPRPRVQ PKPPCGSGDG VSLPPPNKKVAKKPTNKAPH PKQEPQEINF PDDLPGSNTA APVQETLHGC QPVTQEDGKE SRISVQERQ 352hMFE23. MGVLLTQRTL LSLVLALLFP SMASMQVKLE QSGAEVVKPG ASVKLSCKAS CD28.CD2GFNIKDSYMH WLRQGPGQRL EWIGWIDPEN GDTEYAPKFQ GKATFTTDTS CTP321ANTAYLGLSS LRPEDTAVYY CNEGTPTGPY YFDYWGQGTL VTVSSGGGGSGGGGSGGGGS ENVLTQSPSS MSASVGDRVN IACSASSSVS YMHWFQQKPGKSPKLWIYST SNLASGVPSR FSGSGSGTDY SLTISSMQPE DAATYYCQQRSSYPLTFGGG TKLEIKAAAG SGGSGILVKQ SPMLVAYDNA VNLSCKYSYNLFSREFRASL HKGLDSAVEV CVVYGNYSQQ LQVYSKTGFN CDGKLGNESVTFYLQNLYVN QTDIYFCKIE VMYPPPYLDN EKSNGTIIHV KGKHLCPSPLFPGPSKPFWV LVVVGGVLAC YSLLVTVAFI IFWVRSKRSR LLHSDYMNMTPRRPGPTRKH YQPYAPPRDF AAYRSKRRKR NRRRKDEELE IKASRTSTVERGPKPHSTPA AAAQNSVALQ APPPPGHHLQ TPGHRPLPPG HRTREHQQKKRPPPSGTQIH QQKGPPLPRP RVQPKPPCGS GDGVSLPPPN 353 hMFE23.CDMGVLLTQRTL LSLVLALLFP SMASMQVKLE QSGAEVVKPG ASVKLSCKAS 28.GFNIKDSYMH WLRQGPGQRL EWIGWIDPEN GDTEYAPKFQ GKATFTTDTS CD40.CD2ANTAYLGLSS LRPEDTAVYY CNEGTPTGPY YFDYWGQGTL VTVSSGGGGS CTP322GGGGSGGGGS ENVLTQSPSS MSASVGDRVN IACSASSSVS YMHWFQQKPGKSPKLWIYST SNLASGVPSR FSGSGSGTDY SLTISSMQPE DAATYYCQQRSSYPLTFGGG TKLEIKAAAG SGGSGILVKQ SPMLVAYDNA VNLSCKYSYNLFSREFRASL HKGLDSAVEV CVVYGNYSQQ LQVYSKTGFN CDGKLGNESVTFYLQNLYVN QTDIYFCKIE VMYPPPYLDN EKSNGTIIHV KGKHLCPSPLFPGPSKPFWV LVVVGGVLAC YSLLVTVAFI IFWVRSKRSR LLHSDYMNMTPRRPGPTRKH YQPYAPPRDF AAYRSKKVAK KPTNKAPHPK QEPQEINFPDDLPGSNTAAP VQETLHGCQP VTQEDGKESR ISVQERQKRR KRNRRRKDEELEIKASRTST VERGPKPHST PAAAAQNSVA LQAPPPPGHH LQTPGHRPLPPGHRTREHQQ KKRPPPSGTQ IHQQKGPPLP RPRVQPKPPC GSGDGVSLPP PN 354 hMFE23.MGVLLTQRTL LSLVLALLFP SMASMQVKLE QSGAEVVKPG ASVKLSCKAS CD28.CD13GFNIKDSYMH WLRQGPGQRL EWIGWIDPEN GDTEYAPKFQ GKATFTTDTS 7ANTAYLGLSS LRPEDTAVYY CNEGTPTGPY YFDYWGQGTL VTVSSGGGGS CTP323GGGGSGGGGS ENVLTQSPSS MSASVGDRVN IACSASSSVS YMHWFQQKPGKSPKLWIYST SNLASGVPSR FSGSGSGTDY SLTISSMQPE DAATYYCQQRSSYPLTFGGG TKLEIKAAAG SGGSGILVKQ SPMLVAYDNA VNLSCKYSYNLFSREFRASL HKGLDSAVEV CVVYGNYSQQ LQVYSKTGFN CDGKLGNESVTFYLQNLYVN QTDIYFCKIE VMYPPPYLDN EKSNGTIIHV KGKHLCPSPLFPGPSKPFWV LVVVGGVLAC YSLLVTVAFI IFWVRSKRSR LLHSDYMNMTPRRPGPTRKH YQPYAPPRDF AAYRSKRGRK KLLYIFKQPF MRPVQTTQEEDGCSCRFPEE EEGGCEL 355 hMFE23.CDMGVLLTQRTL LSLVLALLFP SMASMQVKLE QSGAEVVKPG ASVKLSCKAS 28.GFNIKDSYMH WLRQGPGQRL EWIGWIDPEN GDTEYAPKFQ GKATFTTDTS CD40.CD13ANTAYLGLSS LRPEDTAVYY CNEGTPTGPY YFDYWGQGTL VTVSSGGGGS 7GGGGSGGGGS ENVLTQSPSS MSASVGDRVN IACSASSSVS YMHWFQQKPG CTP324KSPKLWIYST SNLASGVPSR FSGSGSGTDY SLTISSMQPE DAATYYCQQRSSYPLTFGGG TKLEIKAAAG SGGSGILVKQ SPMLVAYDNA VNLSCKYSYNLFSREFRASL HKGLDSAVEV CVVYGNYSQQ LQVYSKTGFN CDGKLGNESVTFYLQNLYVN QTDIYFCKIE VMYPPPYLDN EKSNGTIIHV KGKHLCPSPLFPGPSKPFWV LVVVGGVLAC YSLLVTVAFI IFWVRSKRSR LLHSDYMNMTPRRPGPTRKH YQPYAPPRDF AAYRSKKVAK KPTNKAPHPK QEPQEINFPDDLPGSNTAAP VQETLHGCQP VTQEDGKESR ISVQERQKRG RKKLLYIFKQPFMRPVQTTQ EEDGCSCRFP EEEEGGCEL 356 hMFE23.CDMGVLLTQRTL LSLVLALLFP SMASMQVKLE QSGAEVVKPG ASVKLSCKAS 28.GFNIKDSYMH WLRQGPGQRL EWIGWIDPEN GDTEYAPKFQ GKATFTTDTS DAP.10ANTAYLGLSS LRPEDTAVYY CNEGTPTGPY YFDYWGQGTL VTVSSGGGGS CTP325GGGGSGGGGS ENVLTQSPSS MSASVGDRVN IACSASSSVS YMHWFQQKPGKSPKLWIYST SNLASGVPSR FSGSGSGTDY SLTISSMQPE DAATYYCQQRSSYPLTFGGG TKLEIKAAAG SGGSGILVKQ SPMLVAYDNA VNLSCKYSYNLFSREFRASL HKGLDSAVEV CVVYGNYSQQ LQVYSKTGFN CDGKLGNESVTFYLQNLYVN QTDIYFCKIE VMYPPPYLDN EKSNGTIIHV KGKHLCPSPLFPGPSKPFWV LVVVGGVLAC YSLLVTVAFI IFWVRSKRSR LLHSDYMNMTPRRPGPTRKH YQPYAPPRDF AAYRSLCARP RRSPAQEDGK VYINMPGRG 357 hMFE23.CDMGVLLTQRTL LSLVLALLFP SMASMQVKLE QSGAEVVKPG ASVKLSCKAS 28.GFNIKDSYMH WLRQGPGQRL EWIGWIDPEN GDTEYAPKFQ GKATFTTDTS CD40.ANTAYLGLSS LRPEDTAVYY CNEGTPTGPY YFDYWGQGTL VTVSSGGGGS DAP10GGGGSGGGGS ENVLTQSPSS MSASVGDRVN IACSASSSVS YMHWFQQKPG CTP326KSPKLWIYST SNLASGVPSR FSGSGSGTDY SLTISSMQPE DAATYYCQQRSSYPLTFGGG TKLEIKAAAG SGGSGILVKQ SPMLVAYDNA VNLSCKYSYNLFSREFRASL HKGLDSAVEV CVVYGNYSQQ LQVYSKTGFN CDGKLGNESVTFYLQNLYVN QTDIYFCKIE VMYPPPYLDN EKSNGTIIHV KGKHLCPSPLFPGPSKPFWV LVVVGGVLAC YSLLVTVAFI IFWVRSKRSR LLHSDYMNMTPRRPGPTRKH YQPYAPPRDF AAYRSKKVAK KPTNKAPHPK QEPQEINFPDDLPGSNTAAP VQETLHGCQP VTQEDGKESR ISVQERQLCA RPRRSPAQED GKVYINMPGR G 358hMFE23.CD MGVLLTQRTL LSLVLALLFP SMASMQVKLE QSGAEVVKPG ASVKLSCKAS 28.GFNIKDSYMH WLRQGPGQRL EWIGWIDPEN GDTEYAPKFQ GKATFTTDTS CD134ANTAYLGLSS LRPEDTAVYY CNEGTPTGPY YFDYWGQGTL VTVSSGGGGS CTP327GGGGSGGGGS ENVLTQSPSS MSASVGDRVN IACSASSSVS YMHWFQQKPGKSPKLWIYST SNLASGVPSR FSGSGSGTDY SLTISSMQPE DAATYYCQQRSSYPLTFGGG TKLEIKAAAG SGGSGILVKQ SPMLVAYDNA VNLSCKYSYNLFSREFRASL HKGLDSAVEV CVVYGNYSQQ LQVYSKTGFN CDGKLGNESVTFYLQNLYVN QTDIYFCKIE VMYPPPYLDN EKSNGTIIHV KGKHLCPSPLFPGPSKPFWV LVVVGGVLAC YSLLVTVAFI IFWVRSKRSR LLHSDYMNMTPRRPGPTRKH YQPYAPPRDF AAYRSALYLL RRDQRLPPDA HKPPGGGSFRTPIQEEQADA HSTLAKI 359 hMFE23.CDMGVLLTQRTL LSLVLALLFP SMASMQVKLE QSGAEVVKPG ASVKLSCKAS 28.GFNIKDSYMH WLRQGPGQRL EWIGWIDPEN GDTEYAPKFQ GKATFTTDTS CD40.ANTAYLGLSS LRPEDTAVYY CNEGTPTGPY YFDYWGQGTL VTVSSGGGGS CD134GGGGSGGGGS ENVLTQSPSS MSASVGDRVN IACSASSSVS YMHWFQQKPG CTP328KSPKLWIYST SNLASGVPSR FSGSGSGTDY SLTISSMQPE DAATYYCQQRSSYPLTFGGG TKLEIKAAAG SGGSGILVKQ SPMLVAYDNA VNLSCKYSYNLFSREFRASL HKGLDSAVEV CVVYGNYSQQ LQVYSKTGFN CDGKLGNESVTFYLQNLYVN QTDIYFCKIE VMYPPPYLDN EKSNGTIIHV KGKHLCPSPLFPGPSKPFWV LVVVGGVLAC YSLLVTVAFI IFWVRSKRSR LLHSDYMNMTPRRPGPTRKH YQPYAPPRDF AAYRSKKVAK KPTNKAPHPK QEPQEINFPDDLPGSNTAAP VQETLHGCQP VTQEDGKESR ISVQERQALY LLRRDQRLPPDAHKPPGGGS FRTPIQEEQA DAHSTLAKI 360 PD1_PD1_SMQIPQAPWPV VWAVLQLGWR PGWFLDSPDR PWNPPTFSPA LLVVTEGDNA CD28TMTFTCSFSNTS ESFVLNWYRM SPSNQTDKLA AFPEDRSQPG QDCRFRVTQL _CD28_PNGRDFHMSV VRARRNDSGT YLCGAISLAP KAQIKESLRA ELRVTERRAE CD40VPTAHPSPSP RPAGQFWVLV VVGGVLACYS LLVTVAFIIF WVRSKRSRLL (monomeric)HSDYMNMTPR RPGPTRKHYQ PYAPPRDFAA YRSKKVAKKP TNKAPHPKQE CTP189PQEINFPDDL PGSNTAAPVQ ETLHGCQPVT QEDGKESRIS VQERQ 361 OSM_MFE23MGVLLTQRTL LSLVLALLFP SMASMQVQLQ QSGAELVRSG TSVKLSCTAS _spCD28GFNIKDSYMH WLRQGPEQGL EWIGWIDPEN GDTEYAPKFQ GKATFTTDTS _CD28_SNTAYLQLSS LTSEDTAVYY CNEGTPTGPY YFDYWGQGTT VTVSSGGGGS CD40GGGGSGGGGS ENVLTQSPAI MSASPGEKVT ITCSASSSVS YMHWFQQKPG (SVQE-TSPKLWIYST SNLASGVPAR FSGSGSGTSY SLTISRMEAE DAATYYCQQR AVQA)SSYPLTFGAG TKLELKRAAA GSGGSGILVK QSPMLVAYDN AVNLSCKYSY CTP195NLFSREFRAS LHKGLDSAVE VCVVYGNYSQ QLQVYSKTGF NCDGKLGNESVTFYLQNLYV NQTDIYFCKI EVMYPPPYLD NEKSNGTIIH VKGKHLCPSPLFPGPSKPFW VLVVVGGVLA CYSLLVTVAF IIFWVRSKRS RLLHSDYMNMTPRRPGPTRK HYQPYAPPRD FAAYRSKKVA KKPTNKAPHP KQEPQEINFPDDLPGSNTAA PVQETLHGCQ PVTQEDGKES RIAVQARQ 362 OSM_MFE23MGVLLTQRTL LSLVLALLFP SMASMQVQLQ QSGAELVRSG TSVKLSCTAS _spCD28GFNIKDSYMH WLRQGPEQGL EWIGWIDPEN GDTEYAPKFQ GKATFTTDTS _CD28_SNTAYLQLSS LTSEDTAVYY CNEGTPTGPY YFDYWGQGTT VTVSSGGGGS CD40GGGGSGGGGS ENVLTQSPAI MSASPGEKVT ITCSASSSVS YMHWFQQKPG (PVQET-TSPKLWIYST SNLASGVPAR FSGSGSGTSY SLTISRMEAE DAATYYCQQR AVAEA)SSYPLTFGAG TKLELKRAAA GSGGSGILVK QSPMLVAYDN AVNLSCKYSY CTP196NLFSREFRAS LHKGLDSAVE VCVVYGNYSQ QLQVYSKTGF NCDGKLGNESVTFYLQNLYV NQTDIYFCKI EVMYPPPYLD NEKSNGTIIH VKGKHLCPSPLFPGPSKPFW VLVVVGGVLA CYSLLVTVAF IIFWVRSKRS RLLHSDYMNMTPRRPGPTRK HYQPYAPPRD FAAYRSKKVA KKPTNKAPHP KQEPQEINFPDDLPGSNTAA AVAEALHGCQ PVTQEDGKES RISVQERQ 363 OSM_MFE23MGVLLTQRTL LSLVLALLFP SMASMQVOLQ QSGAELVRSG TSVKLSCTAS _spCD28GFNIKDSYMH WLRQGPEQGL EWIGWIDPEN GDTEYAPKFQ GKATFTTDTS _CD28_SNTAYLQLSS LTSEDTAVYY CNEGTPTGPY YFDYWGQGTT VTVSSGGGGS CD40GGGGSGGGGS ENVLTQSPAI MSASPGEKVT ITCSASSSVS YMHWFQQKPG (PQEINF-TSPKLWIYST SNLASGVPAR FSGSGSGTSY SLTISRMEAE DAATYYCQQR AQAINF)SSYPLTFGAG TKLELKRAAA GSGGSGILVK QSPMLVAYDN AVNLSCKYSY CTP197NLFSREFRAS LHKGLDSAVE VCVVYGNYSQ QLQVYSKTGF NCDGKLGNESVTFYLQNLYV NQTDIYFCKI EVMYPPPYLD NEKSNGTIIH VKGKHLCPSPLFPGPSKPFW VLVVVGGVLA CYSLLVTVAF IIFWVRSKRS RLLHSDYMNMTPRRPGPTRK HYQPYAPPRD FAAYRSKKVA KKPTNKAPHP KQEAQAINFPDDLPGSNTAA PVQETLHGCQ PVTQEDGKES RISVQERQ 364 OSM_MFE23MGVLLTQRTL LSLVLALLFP SMASMQVQLQ QSGAELVRSG TSVKLSCTAS _spCD28GFNIKDSYMH WLRQGPEQGL EWIGWIDPEN GDTEYAPKFQ GKATFTTDTS _CD28_SNTAYLQLSS LTSEDTAVYY CNEGTPTGPY YFDYWGQGTT VTVSSGGGGS CD40GGGGSGGGGS ENVLTQSPAI MSASPGEKVT ITCSASSSVS YMHWFQQKPG (P227A)TSPKLWIYST SNLASGVPAR FSGSGSGTSY SLTISRMEAE DAATYYCQQR CTP198SSYPLTFGAG TKLELKRAAA GSGGSGILVK QSPMLVAYDN AVNLSCKYSYNLFSREFRAS LHKGLDSAVE VCVVYGNYSQ QLQVYSKTGF NCDGKLGNESVTFYLQNLYV NQTDIYFCKI EVMYPPPYLD NEKSNGTIIH VKGKHLCPSPLFPGPSKPFW VLVVVGGVLA CYSLLVTVAF IIFWVRSKRS RLLHSDYMNMTPRRPGPTRK HYQPYAPPRD FAAYRSKKVA KKPTNKAAHP KQEPQEINFPDDLPGSNTAA PVQETLHGCQ PVTQEDGKES RISVQERQ 365 OSM_MFE23MGVLLTQRTL LSLVLALLFP SMASMQVQLQ QSGAELVRSG TSVKLSCTAS _spCD28_GFNIKDSYMH WLRQGPEQGL EWIGWIDPEN GDTEYAPKFQ GKATFTTDTS CD28SNTAYLQLSS LTSEDTAVYY CNEGTPTGPY YFDYWGQGTT VTVSSGGGGS _CD40GGGGSGGGGS ENVLTQSPAI MSASPGEKVT ITCSASSSVS YMHWFQQKPG (Q263A)TSPKLWIYST SNLASGVPAR FSGSGSGTSY SLTISRMEAE DAATYYCQQR CTP199SSYPLTFGAG TKLELKRAAA GSGGSGILVK QSPMLVAYDN AVNLSCKYSYNLFSREFRAS LHKGLDSAVE VCVVYGNYSQ QLQVYSKTGF NCDGKLGNESVTFYLQNLYV NQTDIYFCKI EVMYPPPYLD NEKSNGTIIH VKGKHLCPSPLFPGPSKPFW VLVVVGGVLA CYSLLVTVAF IIFWVRSKRS RLLHSDYMNMTPRRPGPTRK HYQPYAPPRD FAAYRSKKVA KKPTNKAPHP KQEPQEINFPDDLPGSNTAA PVQETLHGCQ PVTAEDGKES RISVQERQ 366 OSM_MFE23MGVLLTQRTL LSLVLALLFP SMASMQVQLQ QSGAELVRSG TSVKLSCTAS _spCD28GFNIKDSYMH WLRQGPEQGL EWIGWIDPEN GDTEYAPKFQ GKATFTTDTS _CD28SNTAYLQLSS LTSEDTAVYY CNEGTPTGPY YFDYWGQGTT VTVSSGGGGS _CD40GGGGSGGGGS ENVLTQSPAI MSASPGEKVT ITCSASSSVS YMHWFQQKPG _CD40_TSPKLWIYST SNLASGVPAR FSGSGSGTSY SLTISRMEAE DAATYYCQQR CD40SSYPLTFGAG TKLELKRAAA GSGGSGILVK QSPMLVAYDN AVNLSCKYSY CTP200NLFSREFRAS LHKGLDSAVE VCVVYGNYSQ QLQVYSKTGF NCDGKLGNESVTFYLQNLYV NQTDIYFCKI EVMYPPPYLD NEKSNGTIIH VKGKHLCPSPLFPGPSKPFW VLVVVGGVLA CYSLLVTVAF IIFWVRSKRS RLLHSDYMNMTPRRPGPTRK HYQPYAPPRD FAAYRSKKVA KKPTNKAPHP KQEPQEINFPDDLPGSNTAA PVQETLHGCQ PVTAEDGKES RISVQERQKK VAKKPTNKAPHPKQEPQEIN FPDDLPGSNT AAPVQETLHG CQPVTAEDGK ESRISVQERQ 367 OSM_MFE23MGVLLTQRTL LSLVLALLFP SMASMQVQLQ QSGAELVRSG TSVKLSCTAS _spCD28_CGFNIKDSYMH WLRQGPEQGL EWIGWIDPEN GDTEYAPKFQ GKATFTTDTS D28SNTAYLQLSS LTSEDTAVYY CNEGTPTGPY YFDYWGQGTT VTVSSGGGGS (PYAPP-GGGGSGGGGS ENVLTQSPAI MSASPGEKVT ITCSASSSVS YMHWFQQKPG AYAA)TSPKLWIYST SNLASGVPAR FSGSGSGTSY SLTISRMEAE DAATYYCQQR _CD40SSYPLTFGAG TKLELKRAAA GSGGSGILVK QSPMLVAYDN AVNLSCKYSY CTP201NLFSREFRAS LHKGLDSAVE VCVVYGNYSQ QLQVYSKTGF NCDGKLGNESVTFYLQNLYV NQTDIYFCKI EVMYPPPYLD NEKSNGTIIH VKGKHLCPSPLFPGPSKPFW VLVVVGGVLA CYSLLVTVAF IIFWVRSKRS RLLHSDYMNMTPRRPGPTRK HYQAYAAPRD FAAYRSKKVA KKPTNKAPHP KQEPQEINFPDDLPGSNTAA PVQETLHGCQ PVTQEDGKES RISVQERQ 368 OSM_MFE23MGVLLTQRTL LSLVLALLFP SMASMQVOLQ QSGAELVRSG TSVKLSCTAS _spCD28_CGFNIKDSYMH WLRQGPEQGL EWIGWIDPEN GDTEYAPKFQ GKATFTTDTS D28SNTAYLQLSS LTSEDTAVYY CNEGTPTGPY YFDYWGQGTT VTVSSGGGGS (YMNM-GGGGSGGGGS ENVLTQSPAI MSASPGEKVT ITCSASSSVS YMHWFQQKPG FMNM)TSPKLWIYST SNLASGVPAR FSGSGSGTSY SLTISRMEAE DAATYYCQQR _CD40SSYPLTFGAG TKLELKRAAA GSGGSGILVK QSPMLVAYDN AVNLSCKYSY CTP202NLFSREFRAS LHKGLDSAVE VCVVYGNYSQ QLQVYSKTGF NCDGKLGNESVTFYLQNLYV NQTDIYFCKI EVMYPPPYLD NEKSNGTIIH VKGKHLCPSPLFPGPSKPFW VLVVVGGVLA CYSLLVTVAF IIFWVRSKRS RLLHSDFMNMTPRRPGPTRK HYQPYAPPRD FAAYRSKKVA KKPTNKAPHP KQEPQEINFPDDLPGSNTAA PVQETLHGCQ PVTQEDGKES RISVQERQ 369 OSM_MFE23MGVLLTQRTL LSLVLALLFP SMASMQVQLQ QSGAELVRSG TSVKLSCTAS _spCD28_GFNIKDSYMH WLRQGPEQGL EWIGWIDPEN GDTEYAPKFQ GKATFTTDTS CD40SNTAYLQLSS LTSEDTAVYY CNEGTPTGPY YFDYWGQGTT VTVSSGGGGSGGGGSGGGGS ENVLTQSPAI MSASPGEKVT ITCSASSSVS YMHWFQQKPGTSPKLWIYST SNLASGVPAR FSGSGSGTSY SLTISRMEAE DAATYYCQQRSSYPLTFGAG TKLELKRAAA GSGGSGILVK QSPMLVAYDN AVNLSCKYSYNLFSREFRAS LHKGLDSAVE VCVVYGNYSQ QLQVYSKTGF NCDGKLGNESVTFYLQNLYV NQTDIYFCKI EVMYPPPYLD NEKSNGTIIH VKGKHLCPSPLFPGPSKPFW VLVVVGGVLA CYSLLVTVAF IIFWVKKVAK KPTNKAPHPKQEPQEINFPD DLPGSNTAAP VQETLHGCQP VTQEDGKESR ISVQERQ 370 OSM_MFE23MGVLLTQRTL LSLVLALLFP SMASMQVQLQ QSGAELVRSG TSVKLSCTAS _spCD28GFNIKDSYMH WLRQGPEQGL EWIGWIDPEN GDTEYAPKFQ GKATFTTDTS _CD137_SNTAYLQLSS LTSEDTAVYY CNEGTPTGPY YFDYWGQGTT VTVSSGGGGS CD40GGGGSGGGGS ENVLTQSPAI MSASPGEKVT ITCSASSSVS YMHWFQQKPGTSPKLWIYST SNLASGVPAR FSGSGSGTSY SLTISRMEAE DAATYYCQQRSSYPLTFGAG TKLELKRAAA GSGGSGILVK QSPMLVAYDN AVNLSCKYSYNLFSREFRAS LHKGLDSAVE VCVVYGNYSQ QLQVYSKTGF NCDGKLGNESVTFYLQNLYV NQTDIYFCKI EVMYPPPYLD NEKSNGTIIH VKGKHLCPSPLFPGPSKPFW VLVVVGGVLA CYSLLVTVAF IIFWVRFSVV KRGRKKLLYIFKQPFMRPVQ TTQEEDGCSC RFPEEEEGGC EKKVAKKPTN KAPHPKQEPQEINFPDDLPG SNTAAPVQET LHGCQPVTQE DGKESRISVQ ERQ 371 OSM_MFE23MGVLLTQRTL LSLVLALLFP SMASMQVQLQ QSGAELVRSG TSVKLSCTAS _spCD28GFNIKDSYMH WLRQGPEQGL EWIGWIDPEN GDTEYAPKFQ GKATFTTDTS _CD134_SNTAYLQLSS LTSEDTAVYY CNEGTPTGPY YFDYWGQGTT VTVSSGGGGS CD40GGGGSGGGGS ENVLTQSPAI MSASPGEKVT ITCSASSSVS YMHWFQQKPGTSPKLWIYST SNLASGVPAR FSGSGSGTSY SLTISRMEAE DAATYYCQQRSSYPLTFGAG TKLELKRAAA GSGGSGILVK QSPMLVAYDN AVNLSCKYSYNLFSREFRAS LHKGLDSAVE VCVVYGNYSQ QLQVYSKTGF NCDGKLGNESVTFYLQNLYV NQTDIYFCKI EVMYPPPYLD NEKSNGTIIH VKGKHLCPSPLFPGPSKPFW VLVVVGGVLA CYSLLVTVAF IIFWVRRDQR LPPDAHKPPGGGSFRTPIQE EQADAHSTLA KIKKVAKKPT NKAPHPKQEP QEINFPDDLPGSNTAAPVQE TLHGCQPVTQ EDGKESRISV QERQ 372 OSM_MFE23MGVLLTQRTL LSLVLALLFP SMASMQVQLQ QSGAELVRSG TSVKLSCTAS _spCD28GFNIKDSYMH WLRQGPEQGL EWIGWIDPEN GDTEYAPKFQ GKATFTTDTS _CD2_CD40SNTAYLQLSS LTSEDTAVYY CNEGTPTGPY YFDYWGQGTT VTVSSGGGGSGGGGSGGGGS ENVLTQSPAI MSASPGEKVT ITCSASSSVS YMHWFQQKPGTSPKLWIYST SNLASGVPAR FSGSGSGTSY SLTISRMEAE DAATYYCQQRSSYPLTFGAG TKLELKRAAA GSGGSGILVK QSPMLVAYDN AVNLSCKYSYNLFSREFRAS LHKGLDSAVE VCVVYGNYSQ QLQVYSKTGF NCDGKLGNESVTFYLQNLYV NQTDIYFCKI EVMYPPPYLD NEKSNGTIIH VKGKHLCPSPLFPGPSKPFW VLVVVGGVLA CYSLLVTVAF IIFWVKRKKQ RSRRNDEELETRAHRVATEE RGRKPHQIPA STPQNPATSQ HPPPPPGHRS QAPSHRPPPPGHRVQHQPQK RPPAPSGTQV HQQKGPPLPR PRVQPKPPHG AAENSLSPSSNKKVAKKPTN KAPHPKQEPQ EINFPDDLPG SNTAAPVQET LHGCQPVTQE DGKESRISVQ ERQ373 OSM_MFE23 MGVLLTQRTL LSLVLALLFP SMASMQVOLQ QSGAELVRSG TSVKLSCTAS_spCD28 GFNIKDSYMH WLRQGPEQGL EWIGWIDPEN GDTEYAPKFQ GKATFTTDTS _GITR_SNTAYLQLSS LTSEDTAVYY CNEGTPTGPY YFDYWGQGTT VTVSSGGGGS CD40GGGGSGGGGS ENVLTQSPAI MSASPGEKVT ITCSASSSVS YMHWFQQKPGTSPKLWIYST SNLASGVPAR FSGSGSGTSY SLTISRMEAE DAATYYCQQRSSYPLTFGAG TKLELKRAAA GSGGSGILVK QSPMLVAYDN AVNLSCKYSYNLFSREFRAS LHKGLDSAVE VCVVYGNYSQ QLQVYSKTGF NCDGKLGNESVTFYLQNLYV NQTDIYFCKI EVMYPPPYLD NEKSNGTIIH VKGKHLCPSPLFPGPSKPFW VLVVVGGVLA CYSLLVTVAF IIFWVQLGLH IWQLRSQCMWPRETQLLLEV PPSTEDARSC QFPEEERGER SAEEKGRLGD LWVKKVAKKPTNKAPHPKQE PQEINFPDDL PGSNTAAPVQ ETLHGCQPVT QEDGKESRIS VQERQ 374OSM_MFE23 MGVLLTQRTL LSLVLALLFP SMASMQVQLQ QSGAELVRSG TSVKLSCTAS _spCD28GFNIKDSYMH WLRQGPEQGL EWIGWIDPEN GDTEYAPKFQ GKATFTTDTS _CD29_SNTAYLQLSS LTSEDTAVYY CNEGTPTGPY YFDYWGQGTT VTVSSGGGGS CD40GGGGSGGGGS ENVLTQSPAI MSASPGEKVT ITCSASSSVS YMHWFQQKPGTSPKLWIYST SNLASGVPAR FSGSGSGTSY SLTISRMEAE DAATYYCQQRSSYPLTFGAG TKLELKRAAA GSGGSGILVK QSPMLVAYDN AVNLSCKYSYNLFSREFRAS LHKGLDSAVE VCVVYGNYSQ QLQVYSKTGF NCDGKLGNESVTFYLQNLYV NQTDIYFCKI EVMYPPPYLD NEKSNGTIIH VKGKHLCPSPLFPGPSKPFW VLVVVGGVLA CYSLLVTVAF IIFWVKLLMI IHDRREFAKFEKEKMNAKWD TGENPIYKSA VTTVVNPKYE GKKKVAKKPT NKAPHPKQEPQEINFPDDLP GSNTAAPVQE TLHGCQPVTQ EDGKESRISV QERQ 375 OSM_MFE23MGVLLTQRTL LSLVLALLFP SMASMQVQLQ QSGAELVRSG TSVKLSCTAS _spCD28GFNIKDSYMH WLRQGPEQGL EWIGWIDPEN GDTEYAPKFQ GKATFTTDTS _CD150_CDSNTAYLQLSS LTSEDTAVYY CNEGTPTGPY YFDYWGQGTT VTVSSGGGGS 40GGGGSGGGGS ENVLTQSPAI MSASPGEKVT ITCSASSSVS YMHWFQQKPGTSPKLWIYST SNLASGVPAR FSGSGSGTSY SLTISRMEAE DAATYYCQQRSSYPLTFGAG TKLELKRAAA GSGGSGILVK QSPMLVAYDN AVNLSCKYSYNLFSREFRAS LHKGLDSAVE VCVVYGNYSQ QLQVYSKTGF NCDGKLGNESVTFYLQNLYV NQTDIYFCKI EVMYPPPYLD NEKSNGTIIH VKGKHLCPSPLFPGPSKPFW VLVVVGGVLA CYSLLVTVAF IIFWVRRRGK TNHYQTTVEKKSLTIYAQVQ KPGPLQKKLD SFPAQDPCTT IYVAATEPVP ESVQETNSITVYASVTLPES KKVAKKPTNK APHPKQEPQE INFPDDLPGS NTAAPVQETLHGCQPVTQED GKESRISVQE RQ 376 OSM_MFE23MGVLLTQRTL LSLVLALLFP SMASMQVQLQ QSGAELVRSG TSVKLSCTAS _spCD8_GFNIKDSYMH WLRQGPEQGL EWIGWIDPEN GDTEYAPKFQ GKATFTTDTS CD40SNTAYLQLSS LTSEDTAVYY CNEGTPTGPY YFDYWGQGTT VTVSSGGGGS CTP193GGGGSGGGGS ENVLTQSPAI MSASPGEKVT ITCSASSSVS YMHWFQQKPGTSPKLWIYST SNLASGVPAR FSGSGSGTSY SLTISRMEAE DAATYYCQQRSSYPLTFGAG TKLELKRAAA GSGGSGFVPV FLPAKPTTTP APRPPTPAPTIASQPLSLRP EACRPAAGGA VHTRGLDFAC DIYIWAPLAG TCGVLLLSLVITLYCNHRNK KVAKKPTNKA PHPKQEPQEI NFPDDLPGSN TAAPVQETLHGCQPVTQEDG KESRISVQER Q 377 OSM_MFE23MGVLLTQRTL LSLVLALLFP SMASMQVQLQ QSGAELVRSG TSVKLSCTAS _spCD8GFNIKDSYMH WLRQGPEQGL EWIGWIDPEN GDTEYAPKFQ GKATFTTDTS _CD137_SNTAYLQLSS LTSEDTAVYY CNEGTPTGPY YFDYWGQGTT VTVSSGGGGS CD40GGGGSGGGGS ENVLTQSPAI MSASPGEKVT ITCSASSSVS YMHWFQQKPG CTP192TSPKLWIYST SNLASGVPAR FSGSGSGTSY SLTISRMEAE DAATYYCQQRSSYPLTFGAG TKLELKRAAA GSGGSGFVPV FLPAKPTTTP APRPPTPAPTIASQPLSLRP EACRPAAGGA VHTRGLDFAC DIYIWAPLAG TCGVLLLSLVITLYCNHRNR FSVVKRGRKK LLYIFKQPFM RPVQTTQEED GCSCRFPEEEEGGGEKKVAK KPTNKAPHPK QEPQEINFPD DLPGSNTAAP VQETLHGCQPVTQEDGKESR ISVQERQ 378 OSM_MFE23MGVLLTQRTL LSLVLALLFP SMASMQVOLQ QSGAELVRSG TSVKLSCTAS _spCD8GFNIKDSYMH WLRQGPEQGL EWIGWIDPEN GDTEYAPKFQ GKATFTTDTS _CD134_CDSNTAYLQLSS LTSEDTAVYY CNEGTPTGPY YFDYWGQGTT VTVSSGGGGS 40GGGGSGGGGS ENVLTQSPAI MSASPGEKVT ITCSASSSVS YMHWFQQKPGTSPKLWIYST SNLASGVPAR FSGSGSGTSY SLTISRMEAE DAATYYCQQRSSYPLTFGAG TKLELKRAAA GSGGSGFVPV FLPAKPTTTP APRPPTPAPTIASQPLSLRP EACRPAAGGA VHTRGLDFAC DIYIWAPLAG TCGVLLLSLVITLYCNHRNR RDQRLPPDAH KPPGGGSFRT PIQEEQADAH STLAKIKKVAKKPTNKAPHP KQEPQEINFP DDLPGSNTAA PVQETLHGCQ PVTQEDGKSR ISVQERQ 379OSM_MFE23 MGVLLTQRTL LSLVLALLFP SMASMQVQLQ QSGAELVRSG TSVKLSCTAS _spCD8GFNIKDSYMH WLRQGPEQGL EWIGWIDPEN GDTEYAPKFQ GKATFTTDTS _CD2_CD40SNTAYLQLSS LTSEDTAVYY CNEGTPTGPY YFDYWGQGTT VTVSSGGGGS CTP191GGGGSGGGGS ENVLTQSPAI MSASPGEKVT ITCSASSSVS YMHWFQQKPGTSPKLWIYST SNLASGVPAR FSGSGSGTSY SLTISRMEAE DAATYYCQQRSSYPLTFGAG TKLELKRAAA GSGGSGFVPV FLPAKPTTTP APRPPTPAPTIASQPLSLRP EACRPAAGGA VHTRGLDFAC DIYIWAPLAG TCGVLLLSLVITLYCNHRNK RKKQRSRRND EELETRAHRV ATEERGRKPH QIPASTPQNPATSQHPPPPP GHRSQAPSHR PPPPGHRVQH QPQKRPPAPS GTQVHQQKGPPLPRPRVQPK PPHGAAENSL SPSSNKKVAK KPTNKAPHPK QEPQEINFPDDLPGSNTAAP VQETLHGCQP VTQEDGKESR ISVQERQ 380 OSM_MFE23MGVLLTQRTL LSLVLALLFP SMASMQVQLQ QSGAELVRSG TSVKLSCTAS _spCD8GFNIKDSYMH WLRQGPEQGL EWIGWIDPEN GDTEYAPKFQ GKATFTTDTS _GITR_CD4SNTAYLQLSS LTSEDTAVYY CNEGTPTGPY YFDYWGQGTT VTVSSGGGGS 0GGGGSGGGGS ENVLTQSPAI MSASPGEKVT ITCSASSSVS YMHWFQQKPGTSPKLWIYST SNLASGVPAR FSGSGSGTSY SLTISRMEAE DAATYYCQQRSSYPLTFGAG TKLELKRAAA GSGGSGFVPV FLPAKPTTTP APRPPTPAPTIASQPLSLRP EACRPAAGGA VHTRGLDFAC DIYIWAPLAG TCGVLLLSLVITLYCNHRNQ LGLHIWQLRS QCMWPRETQL LLEVPPSTED ARSCQFPEEERGERSAEEKG RLGDLWVKKV AKKPTNKAPH PKQEPQEINF PDDLPGSNTAAPVQETLHGC QPVTQEDGKE SRISVQERQ 381 OSM_MFE23MGVLLTQRTL LSLVLALLFP SMASMQVQLQ QSGAELVRSG TSVKLSCTAS _spCD8GFNIKDSYMH WLRQGPEQGL EWIGWIDPEN GDTEYAPKFQ GKATFTTDTS _CD29_SNTAYLQLSS LTSEDTAVYY CNEGTPTGPY YFDYWGQGTT VTVSSGGGGS CD40GGGGSGGGGS ENVLTQSPAI MSASPGEKVT ITCSASSSVS YMHWFQQKPGTSPKLWIYST SNLASGVPAR FSGSGSGTSY SLTISRMEAE DAATYYCQQRSSYPLTFGAG TKLELKRAAA GSGGSGFVPV FLPAKPTTTP APRPPTPAPTIASQPLSLRP EACRPAAGGA VHTRGLDFAC DIYIWAPLAG TCGVLLLSLVITLYCNHRNK LLMIIHDRRE FAKFEKEKMN AKWDTGENPI YKSAVTTVVNPKYEGKKKVA KKPTNKAPHP KQEPQEINFP DDLPGSNTAA PVQETLHGCQPVTQEDGKES RISVQERQ 382 OSM_MFE23MGVLLTQRTL LSLVLALLFP SMASMQVQLQ QSGAELVRSG TSVKLSCTAS _spCD8GFNIKDSYMH WLRQGPEQGL EWIGWIDPEN GDTEYAPKFQ GKATFTTDTS _CD150_CDSNTAYLQLSS LTSEDTAVYY CNEGTPTGPY YFDYWGQGTT VTVSSGGGGS 40GGGGSGGGGS ENVLTQSPAI MSASPGEKVT ITCSASSSVS YMHWFQQKPGTSPKLWIYST SNLASGVPAR FSGSGSGTSY SLTISRMEAE DAATYYCQQRSSYPLTFGAG TKLELKRAAA GSGGSGFVPV FLPAKPTTTP APRPPTPAPTIASQPLSLRP EACRPAAGGA VHTRGLDFAC DIYIWAPLAG TCGVLLLSLVITLYCNHRNR RRGKTNHYQT TVEKKSLTIY AQVQKPGPLQ KKLDSFPAQDPCTTIYVAAT EPVPESVQET NSITVYASVT LPESKKVAKK PTNKAPHPKQEPQEINFPDD LPGSNTAAPV QETLHGCQPV TQEDGKESRI SVQERQ 383 OSM_MFE23MGVLLTQRTL LSLVLALLFP SMASMQVOLQ QSGAELVRSG TSVKLSCTAS _spIG4GFNIKDSYMH WLRQGPEQGL EWIGWIDPEN GDTEYAPKFQ GKATFTTDTS _CD28_SNTAYLQLSS LTSEDTAVYY CNEGTPTGPY YFDYWGQGTT VTVSSGGGGS CD40GGGGSGGGGS ENVLTQSPAI MSASPGEKVT ITCSASSSVS YMHWFQQKPG CTP203TSPKLWIYST SNLASGVPAR FSGSGSGTSY SLTISRMEAE DAATYYCQQRSSYPLTFGAG TKLELKRAAA GSGGSGESKY GPPCPSCPAP EFLGGPSVFLFPPKPKDTLM ISRTPEVTCV VVDVSQEDPE VQFNWYVDGV EVHNAKTKPREEQFNSTYRV VSVLTVLHQD WLNGKEYKCK VSNKGLPSSI EKTISKAKGQPREPQVYTLP PSQEEMTKNQ VSLTCLVKGF YPSDIAVEWE SNGQPENNYKTTPPVLDSDG SFFLYSRLTV DKSRWQEGNV FSCSVMHEAL HNHYTQKSLSLSLGKMFWVL VVVGGVLACY SLLVTVAFII FWVRSKRSRL LHSDYMNMTPRRPGPTRKHY QPYAPPRDFA AYRSKKVAKK PTNKAPHPKQ EPQEINFPDDLPGSNTAAPV QETLHGCQPV TQEDGKESRI SVQERQ 384 OSM_MFE23MGVLLTQRTL LSLVLALLFP SMASMQVQLQ QSGAELVRSG TSVKLSCTAS _spIG4_GFNIKDSYMH WLRQGPEQGL EWIGWIDPEN GDTEYAPKFQ GKATFTTDTS CD40SNTAYLQLSS LTSEDTAVYY CNEGTPTGPY YFDYWGQGTT VTVSSGGGGSGGGGSGGGGS ENVLTQSPAI MSASPGEKVT ITCSASSSVS YMHWFQQKPGTSPKLWIYST SNLASGVPAR FSGSGSGTSY SLTISRMEAE DAATYYCQQRSSYPLTFGAG TKLELKRAAA GSGGSGESKY GPPCPSCPAP EFLGGPSVFLFPPKPKDTLM ISRTPEVTCV VVDVSQEDPE VQFNWYVDGV EVHNAKTKPREEQFNSTYRV VSVLTVLHQD WLNGKEYKCK VSNKGLPSSI EKTISKAKGQPREPQVYTLP PSQEEMTKNQ VSLTCLVKGF YPSDIAVEWE SNGQPENNYKTTPPVLDSDG SFFLYSRLTV DKSRWQEGNV FSCSVMHEAL HNHYTQKSLSLSLGKMFWVL VVVGGVLACY SLLVTVAFII FWVKKVAKKP TNKAPHPKQEPQEINFPDDL PGSNTAAPVQ ETLHGCQPVT QEDGKESRIS VQERQ 385 OSM_MFE23MGVLLTQRTL LSLVLALLFP SMASMQVQLQ QSGAELVRSG TSVKLSCTAS _spIG4GFNIKDSYMH WLRQGPEQGL EWIGWIDPEN GDTEYAPKFQ GKATFTTDTS _CD137_CDSNTAYLQLSS LTSEDTAVYY CNEGTPTGPY YFDYWGQGTT VTVSSGGGGS 40GGGGSGGGGS ENVLTQSPAI MSASPGEKVT ITCSASSSVS YMHWFQQKPGTSPKLWIYST SNLASGVPAR FSGSGSGTSY SLTISRMEAE DAATYYCQQRSSYPLTFGAG TKLELKRAAA GSGGSGESKY GPPCPSCPAP EFLGGPSVFLFPPKPKDTLM ISRTPEVTCV VVDVSQEDPE VQFNWYVDGV EVHNAKTKPREEQFNSTYRV VSVLTVLHQD WLNGKEYKCK VSNKGLPSSI EKTISKAKGQPREPQVYTLP PSQEEMTKNQ VSLTCLVKGF YPSDIAVEWE SNGQPENNYKTTPPVLDSDG SFFLYSRLTV DKSRWQEGNV FSCSVMHEAL HNHYTQKSLSLSLGKMFWVL VVVGGVLACY SLLVTVAFII FWVKKVAKKP TNKAPHPKQEPQEINFPDDL PGSNTAAPVQ ETLHGCQPVT QEDGKESRIS VQERQ 386 OSM_MFE23MGVLLTQRTL LSLVLALLFP SMASMQVQLQ QSGAELVRSG TSVKLSCTAS _spIG4GFNIKDSYMH WLRQGPEQGL EWIGWIDPEN GDTEYAPKFQ GKATFTTDTS _CD134_SNTAYLQLSS LTSEDTAVYY CNEGTPTGPY YFDYWGQGTT VTVSSGGGGS CD40GGGGSGGGGS ENVLTQSPAI MSASPGEKVT ITCSASSSVS YMHWFQQKPGTSPKLWIYST SNLASGVPAR FSGSGSGTSY SLTISRMEAE DAATYYCQQRSSYPLTFGAG TKLELKRAAA GSGGSGESKY GPPCPSCPAP EFLGGPSVFLFPPKPKDTLM ISRTPEVTCV VVDVSQEDPE VQFNWYVDGV EVHNAKTKPREEQFNSTYRV VSVLTVLHQD WLNGKEYKCK VSNKGLPSSI EKTISKAKGQPREPQVYTLP PSQEEMTKNQ VSLTCLVKGF YPSDIAVEWE SNGQPENNYKTTPPVLDSDG SFFLYSRLTV DKSRWQEGNV FSCSVMHEAL HNHYTQKSLSLSLGKMFWVL VVVGGVLACY SLLVTVAFII FWVRRDQRLP PDAHKPPGGGSFRTPIQEEQ ADAHSTLAKI KKVAKKPTNK APHPKQEPQE INFPDDLPGSNTAAPVQETL HGCQPVTQED GKESRISVQE RQ 387 OSM_MFE23MGVLLTQRTL LSLVLALLFP SMASMQVOLQ QSGAELVRSG TSVKLSCTAS _spIG4GFNIKDSYMH WLRQGPEQGL EWIGWIDPEN GDTEYAPKFQ GKATFTTDTS _CD2_SNTAYLQLSS LTSEDTAVYY CNEGTPTGPY YFDYWGQGTT VTVSSGGGGS CD40GGGGSGGGGS ENVLTQSPAI MSASPGEKVT ITCSASSSVS YMHWFQQKPGTSPKLWIYST SNLASGVPAR FSGSGSGTSY SLTISRMEAE DAATYYCQQRSSYPLTFGAG TKLELKRAAA GSGGSGESKY GPPCPSCPAP EFLGGPSVFLFPPKPKDTLM ISRTPEVTCV VVDVSQEDPE VQFNWYVDGV EVHNAKTKPREEQFNSTYRV VSVLTVLHQD WLNGKEYKCK VSNKGLPSSI EKTISKAKGQPREPQVYTLP PSQEEMTKNQ VSLTCLVKGF YPSDIAVEWE SNGQPENNYKTTPPVLDSDG SFFLYSRLTV DKSRWQEGNV FSCSVMHEAL HNHYTQKSLSLSLGKMFWVL VVVGGVLACY SLLVTVAFII FWVKRKKQRS RRNDEELETRAHRVATEERG RKPHQIPAST PQNPATSQHP PPPPGHRSQA PSHRPPPPGHRVQHQPQKRP PAPSGTQVHQ QKGPPLPRPR VQPKPPHGAA ENSLSPSSNKKVAKKPTNKA PHPKQEPQEI NFPDDLPGSN TAAPVQETLH GCQPVTQEDG KESRISVQER Q 388OSM_MFE23 MGVLLTQRTL LSLVLALLFP SMASMQVQLQ QSGAELVRSG TSVKLSCTAS _spIG4GFNIKDSYMH WLRQGPEQGL EWIGWIDPEN GDTEYAPKFQ GKATFTTDTS _GITR_CD4SNTAYLQLSS LTSEDTAVYY CNEGTPTGPY YFDYWGQGTT VTVSSGGGGS 0GGGGSGGGGS ENVLTQSPAI MSASPGEKVT ITCSASSSVS YMHWFQQKPGTSPKLWIYST SNLASGVPAR FSGSGSGTSY SLTISRMEAE DAATYYCQQRSSYPLTFGAG TKLELKRAAA GSGGSGESKY GPPCPSCPAP EFLGGPSVFLFPPKPKDTLM ISRTPEVTCV VVDVSQEDPE VQFNWYVDGV EVHNAKTKPREEQFNSTYRV VSVLTVLHQD WLNGKEYKCK VSNKGLPSSI EKTISKAKGQPREPQVYTLP PSQEEMTKNQ VSLTCLVKGF YPSDIAVEWE SNGQPENNYKTTPPVLDSDG SFFLYSRLTV DKSRWQEGNV FSCSVMHEAL HNHYTQKSLSLSLGKMFWVL VVVGGVLACY SLLVTVAFII FWVQLGLHIW QLRSQCMWPRETQLLLEVPP STEDARSCQF PEEERGERSA EEKGRLGDLW VKKVAKKPTNKAPHPKQEPQ EINFPDDLPG SNTAAPVQET LHGCQPVTQE DGKESRISVQ ERQ 389 OSM_MFE23MGVLLTQRTL LSLVLALLFP SMASMQVQLQ QSGAELVRSG TSVKLSCTAS _spIG4GFNIKDSYMH WLRQGPEQGL EWIGWIDPEN GDTEYAPKFQ GKATFTTDTS _CD29_SNTAYLQLSS LTSEDTAVYY CNEGTPTGPY YFDYWGQGTT VTVSSGGGGS CD40GGGGSGGGGS ENVLTQSPAI MSASPGEKVT ITCSASSSVS YMHWFQQKPGTSPKLWIYST SNLASGVPAR FSGSGSGTSY SLTISRMEAE DAATYYCQQRSSYPLTFGAG TKLELKRAAA GSGGSGESKY GPPCPSCPAP EFLGGPSVFLFPPKPKDTLM ISRTPEVTCV VVDVSQEDPE VQFNWYVDGV EVHNAKTKPREEQFNSTYRV VSVLTVLHQD WLNGKEYKCK VSNKGLPSSI EKTISKAKGQPREPQVYTLP PSQEEMTKNQ VSLTCLVKGF YPSDIAVEWE SNGQPENNYKTTPPVLDSDG SFFLYSRLTV DKSRWQEGNV FSCSVMHEAL HNHYTQKSLSLSLGKMFWVL VVVGGVLACY SLLVTVAFII FWVKLLMIIH DRREFAKFEKEKMNAKWDTG ENPIYKSAVT TVVNPKYEGK KKVAKKPTNK APHPKQEPQEINFPDDLPGS NTAAPVQETL HGCQPVTQED GKESRISVQE RQ 390 OSM_MFE23MGVLLTQRTL LSLVLALLFP SMASMQVQLQ QSGAELVRSG TSVKLSCTAS _spIG4GFNIKDSYMH WLRQGPEQGL EWIGWIDPEN GDTEYAPKFQ GKATFTTDTS _CD150_SNTAYLQLSS LTSEDTAVYY CNEGTPTGPY YFDYWGQGTT VTVSSGGGGS CD40GGGGSGGGGS ENVLTQSPAI MSASPGEKVT ITCSASSSVS YMHWFQQKPGTSPKLWIYST SNLASGVPAR FSGSGSGTSY SLTISRMEAE DAATYYCQQRSSYPLTFGAG TKLELKRAAA GSGGSGESKY GPPCPSCPAP EFLGGPSVFLFPPKPKDTLM ISRTPEVTCV VVDVSQEDPE VQFNWYVDGV EVHNAKTKPREEQFNSTYRV VSVLTVLHQD WLNGKEYKCK VSNKGLPSSI EKTISKAKGQPREPQVYTLP PSQEEMTKNQ VSLTCLVKGF YPSDIAVEWE SNGQPENNYKTTPPVLDSDG SFFLYSRLTV DKSRWQEGNV FSCSVMHEAL HNHYTQKSLSLSLGKMFWVL VVVGGVLACY SLLVTVAFII FWVRRRGKTN HYQTTVEKKSLTIYAQVQKP GPLQKKLDSF PAQDPCTTIY VAATEPVPES VQETNSITVYASVTLPESKK VAKKPTNKAP HPKQEPQEIN FPDDLPGSNT AAPVQETLHGCQPVTQEDGK ESRISVQERQ 391 OSM_MFE23MGVLLTQRTL LSLVLALLFP SMASMQVOLQ QSGAELVRSG TSVKLSCTAS _spIG4GFNIKDSYMH WLRQGPEQGL EWIGWIDPEN GDTEYAPKFQ GKATFTTDTS _CD40_SNTAYLQLSS LTSEDTAVYY CNEGTPTGPY YFDYWGQGTT VTVSSGGGGS tandemGGGGSGGGGS ENVLTQSPAI MSASPGEKVT ITCSASSSVS YMHWFQQKPGTSPKLWIYST SNLASGVPAR FSGSGSGTSY SLTISRMEAE DAATYYCQQRSSYPLTFGAG TKLELKRAAA GSGGSGESKY GPPCPSCPAP EFLGGPSVFLFPPKPKDTLM ISRTPEVTCV VVDVSQEDPE VQFNWYVDGV EVHNAKTKPREEQFNSTYRV VSVLTVLHQD WLNGKEYKCK VSNKGLPSSI EKTISKAKGQPREPQVYTLP PSQEEMTKNQ VSLTCLVKGF YPSDIAVEWE SNGQPENNYKTTPPVLDSDG SFFLYSRLTV DKSRWQEGNV FSCSVMHEAL HNHYTQKSLSLSLGKMFWVL VVVGGVLACY SLLVTVAFII FWVAKKPTNK APHPKQEPQEINFPDDLPGS NTAAPVQETL HGCQPVTQED GKESRISVQE RQKKVAKKPTNKAPHPKQEP QEINFPDDLP GSNTAAPVQE TLHGCQPVTQ EDGKESRISV QERQKKVA 392OSM_MFE23 MGVLLTQRTL LSLVLALLFP SMASMQVQLQ QSGAELVRSG TSVKLSCTAS _spIG4GFNIKDSYMH WLRQGPEQGL EWIGWIDPEN GDTEYAPKFQ GKATFTTDTS _CD40_SNTAYLQLSS LTSEDTAVYY CNEGTPTGPY YFDYWGQGTT VTVSSGGGGS P227AGGGGSGGGGS ENVLTQSPAI MSASPGEKVT ITCSASSSVS YMHWFQQKPGTSPKLWIYST SNLASGVPAR FSGSGSGTSY SLTISRMEAE DAATYYCQQRSSYPLTFGAG TKLELKRAAA GSGGSGESKY GPPCPSCPAP EFLGGPSVFLFPPKPKDTLM ISRTPEVTCV VVDVSQEDPE VQFNWYVDGV EVHNAKTKPREEQFNSTYRV VSVLTVLHQD WLNGKEYKCK VSNKGLPSSI EKTISKAKGQPREPQVYTLP PSQEEMTKNQ VSLTCLVKGF YPSDIAVEWE SNGQPENNYKTTPPVLDSDG SFFLYSRLTV DKSRWQEGNV FSCSVMHEAL HNHYTQKSLSLSLGKMFWVL VVVGGVLACY SLLVTVAFII FWVKKVAKKP TNKAAHPKQEPQEINFPDDL PGSNTAAPVQ ETLHGCQPVT QEDGKESRIS VQERQ 393 OSM_MFE23MGVLLTQRTL LSLVLALLFP SMASMQVQLQ QSGAELVRSG TSVKLSCTAS _PD1GFNIKDSYMH WLRQGPEQGL EWIGWIDPEN GDTEYAPKFQ GKATFTTDTS _sCD28TM_SNTAYLQLSS LTSEDTAVYY CNEGTPTGPY YFDYWGQGTT VTVSSGGGGS CD28GGGGSGGGGS ENVLTQSPAI MSASPGEKVT ITCSASSSVS YMHWFQQKPG _CD40TSPKLWIYST SNLASGVPAR FSGSGSGTSY SLTISRMEAE DAATYYCQQR CTP204SSYPLTFGAG TKLELKRAAA GSGGSGRPGW FLDSPDRPWN PPTFSPALLVVTEGDNATFT CSFSNTSESF VLNWYRMSPS NQTDKLAAFP EDRSQPGQDCRFRVTQLPNG RDFHMSVVRA RRNDSGTYLC GAISLAPKAQ IKESLRAELRVTERRAEVPT AHCPSPLFPG PSKPFWVLVV VGGVLACYSL LVTVAFIIFWVRSKRSRLLH SDYMNMTPRR PGPTRKHYQP YAPPRDFAAY RSKKVAKKPTNKAPHPKQEP QEINFPDDLP GSNTAAPVQE TLHGCQPVTQ EDGKESRISV QERQ 394OSM_MFE23 MGVLLTQRTL LSLVLALLFP SMASMQVQLQ QSGAELVRSG TSVKLSCTAS _PD1GFNIKDSYMH WLRQGPEQGL EWIGWIDPEN GDTEYAPKFQ GKATFTTDTS _sCD28TM_SNTAYLQLSS LTSEDTAVYY CNEGTPTGPY YFDYWGQGTT VTVSSGGGGS CD40GGGGSGGGGS ENVLTQSPAI MSASPGEKVT ITCSASSSVS YMHWFQQKPGTSPKLWIYST SNLASGVPAR FSGSGSGTSY SLTISRMEAE DAATYYCQQRSSYPLTFGAG TKLELKRAAA GSGGSGRPGW FLDSPDRPWN PPTFSPALLVVTEGDNATFT CSFSNTSESF VLNWYRMSPS NQTDKLAAFP EDRSQPGQDCRFRVTQLPNG RDFHMSVVRA RRNDSGTYLC GAISLAPKAQ IKESLRAELRVTERRAEVPT AHCPSPLFPG PSKPFWVLVV VGGVLACYSL LVTVAFIIFWVKKVAKKPTN KAPHPKQEPQ EINFPDDLPG SNTAAPVQET LHGCQPVTQE DGKESRISVQ ERQ395 OSM_MFE23 MGVLLTQRTL LSLVLALLFP SMASMQVOLQ QSGAELVRSG TSVKLSCTAS_TIGIT GFNIKDSYMH WLRQGPEQGL EWIGWIDPEN GDTEYAPKFQ GKATFTTDTS _sCD28TM_SNTAYLQLSS LTSEDTAVYY CNEGTPTGPY YFDYWGQGTT VTVSSGGGGS CD28GGGGSGGGGS ENVLTQSPAI MSASPGEKVT ITCSASSSVS YMHWFQQKPG _CD40TSPKLWIYST SNLASGVPAR FSGSGSGTSY SLTISRMEAE DAATYYCQQRSSYPLTFGAG TKLELKRAAA GSGGSGMMTG TIETTGNISA EKGGSIILQCHLSSTTAQVT QVNWEQQDQL LAICNADLGW HISPSFKDRV APGPGLGLTLQSLTVNDTGE YFCIYHTYPD GTYTGRIFLE VLESSVAEHG ARFQIPFWVLVVVGGVLACY SLLVTVAFII FWVRSKRSRL LHSDYMNMTP RRPGPTRKHYQPYAPPRDFA AYRSKKVAKK PTNKAPHPKQ EPQEINFPDD LPGSNTAAPVQETLHGCQPV TQEDGKESRI SVQERQ 396 OSM_MFE23MGVLLTQRTL LSLVLALLFP SMASMQVQLQ QSGAELVRSG TSVKLSCTAS _TIGITGFNIKDSYMH WLRQGPEQGL EWIGWIDPEN GDTEYAPKFQ GKATFTTDTS _sCD28TM_SNTAYLQLSS LTSEDTAVYY CNEGTPTGPY YFDYWGQGTT VTVSSGGGGS CD40GGGGSGGGGS ENVLTQSPAI MSASPGEKVT ITCSASSSVS YMHWFQQKPGTSPKLWIYST SNLASGVPAR FSGSGSGTSY SLTISRMEAE DAATYYCQQRSSYPLTFGAG TKLELKRAAA GSGGSGMMTG TIETTGNISA EKGGSIILQCHLSSTTAQVT QVNWEQQDQL LAICNADLGW HISPSFKDRV APGPGLGLTLQSLTVNDTGE YFCIYHTYPD GTYTGRIFLE VLESSVAEHG ARFQIPFWVLVVVGGVLACY SLLVTVAFII FWVKKVAKKP TNKAPHPKQE PQEINFPDDLPGSNTAAPVQ ETLHGCQPVT QEDGKESRIS VQERQ 397 OSM_MOV19MGVLLTQRTL LSLVLALLFP SMASMQVQLQ QSGAELVKPG ASVKISCKAS _spCD28GYSFTGYFMN WVKQSHGKSL EWIGRIHPYD GDTFYNQNFK DKATLTVDKS _CD28_SNTAHMELLS LTSEDFAVYY CTRYDGSRAM DYWGQGTTVT VSSGGGGSGG CD40GGSGGGGSDI ELTQSPASLA VSLGQRAIIS CKASQSVSFA GTSLMHWYHQKPGQQPKLLI YRASNLEAGV PTRFSGSGSK TDFTLNIHPV EEEDAATYYCQQSREYPYTF GGGTKLEIKA AAGSGGSGIL VKQSPMLVAY DNAVNLSCKYSYNLFSREFR ASLHKGLDSA VEVCVVYGNY SQQLQVYSKT GFNCDGKLGNESVTFYLQNL YVNQTDIYFC KIEVMYPPPY LDNEKSNGTI IHVKGKHLCPSPLFPGPSKP FWVLVVVGGV LACYSLLVTV AFIIFWVRSK RSRLLHSDYMNMTPRRPGPT RKHYQPYAPP RDFAAYRSKK VAKKPTNKAP HPKQEPQEINFPDDLPGSNT AAPVQETLHG CQPVTQEDGK ESRISVQERQ 398 OSM_MOV19MGVLLTQRTL LSLVLALLFP SMASMQVQLQ QSGAELVKPG ASVKISCKAS _spCD28_GYSFTGYFMN WVKQSHGKSL EWIGRIHPYD GDTFYNQNFK DKATLTVDKS CD40SNTAHMELLS LTSEDFAVYY CTRYDGSRAM DYWGQGTTVT VSSGGGGSGGGGSGGGGSDI ELTQSPASLA VSLGQRAIIS CKASQSVSFA GTSLMHWYHQKPGQQPKLLI YRASNLEAGV PTRFSGSGSK TDFTLNIHPV EEEDAATYYCQQSREYPYTF GGGTKLEIKA AAGSGGSGIL VKQSPMLVAY DNAVNLSCKYSYNLFSREFR ASLHKGLDSA VEVCVVYGNY SQQLQVYSKT GFNCDGKLGNESVTFYLQNL YVNQTDIYFC KIEVMYPPPY LDNEKSNGTI IHVKGKHLCPSPLFPGPSKP FWVLVVVGGV LACYSLLVTV AFIIFWVKKV AKKPTNKAPHPKQEPQEINF PDDLPGSNTA APVQETLHGC QPVTQEDGKE SRISVQERQ 399 OSM_MOV19MGVLLTQRTL LSLVLALLFP SMASMQVQLQ QSGAELVKPG ASVKISCKAS _spCD28GYSFTGYFMN WVKQSHGKSL EWIGRIHPYD GDTFYNQNFK DKATLTVDKS _CD137_SNTAHMELLS LTSEDFAVYY CTRYDGSRAM DYWGQGTTVT VSSGGGGSGG CD40GGSGGGGSDI ELTQSPASLA VSLGQRAIIS CKASQSVSFA GTSLMHWYHQKPGQQPKLLI YRASNLEAGV PTRFSGSGSK TDFTLNIHPV EEEDAATYYCQQSREYPYTF GGGTKLEIKA AAGSGGSGIL VKQSPMLVAY DNAVNLSCKYSYNLFSREFR ASLHKGLDSA VEVCVVYGNY SQQLQVYSKT GFNCDGKLGNESVTFYLQNL YVNQTDIYFC KIEVMYPPPY LDNEKSNGTI IHVKGKHLCPSPLFPGPSKP FWVLVVVGGV LACYSLLVTV AFIIFWVRFS VVKRGRKKLLYIFKQPFMRP VQTTQEEDGC SCRFPEEEEG GCEKKVAKKP TNKAPHPKQEPQEINFPDDL PGSNTAAPVQ ETLHGCQPVT QEDGKESRIS VQERQ 400 OSM_MOV19MGVLLTQRTL LSLVLALLFP SMASMQVOLQ QSGAELVKPG ASVKISCKAS _spCD28GYSFTGYFMN WVKQSHGKSL EWIGRIHPYD GDTFYNQNFK DKATLTVDKS _CD134_CDSNTAHMELLS LTSEDFAVYY CTRYDGSRAM DYWGQGTTVT VSSGGGGSGG 40GGSGGGGSDI ELTQSPASLA VSLGQRAIIS CKASQSVSFA GTSLMHWYHQKPGQQPKLLI YRASNLEAGV PTRFSGSGSK TDFTLNIHPV EEEDAATYYCQQSREYPYTF GGGTKLEIKA AAGSGGSGIL VKQSPMLVAY DNAVNLSCKYSYNLFSREFR ASLHKGLDSA VEVCVVYGNY SQQLQVYSKT GFNCDGKLGNESVTFYLQNL YVNQTDIYFC KIEVMYPPPY LDNEKSNGTI IHVKGKHLCPSPLFPGPSKP FWVLVVVGGV LACYSLLVTV AFIIFWVRRD QRLPPDAHKPPGGGSFRTPI QEEQADAHST LAKIKKVAKK PTNKAPHPKQ EPQEINFPDDLPGSNTAAPV QETLHGCQPV TQEDGKESRI SVQERQ 401 OSM_MOV19MGVLLTQRTL LSLVLALLFP SMASMQVQLQ QSGAELVKPG ASVKISCKAS _spCD28GYSFTGYFMN WVKQSHGKSL EWIGRIHPYD GDTFYNQNFK DKATLTVDKS _CD2_CD40SNTAHMELLS LTSEDFAVYY CTRYDGSRAM DYWGQGTTVT VSSGGGGSGGGGSGGGGSDI ELTQSPASLA VSLGQRAIIS CKASQSVSFA GTSLMHWYHQKPGQQPKLLI YRASNLEAGV PTRFSGSGSK TDFTLNIHPV EEEDAATYYCQQSREYPYTF GGGTKLEIKA AAGSGGSGIL VKQSPMLVAY DNAVNLSCKYSYNLFSREFR ASLHKGLDSA VEVCVVYGNY SQQLQVYSKT GFNCDGKLGNESVTFYLQNL YVNQTDIYFC KIEVMYPPPY LDNEKSNGTI IHVKGKHLCPSPLFPGPSKP FWVLVVVGGV LACYSLLVTV AFIIFWVKRK KQRSRRNDEELETRAHRVAT EERGRKPHQI PASTPQNPAT SQHPPPPPGH RSQAPSHRPPPPGHRVQHQP QKRPPAPSGT QVHQQKGPPL PRPRVQPKPP HGAAENSLSPSSNKKVAKKP TNKAPHPKQE PQEINFPDDL PGSNTAAPVQ ETLHGCQPVT QEDGKESRIS VQERQ402 OSM_MOV19 MGVLLTQRTL LSLVLALLFP SMASMQVQLQ QSGAELVKPG ASVKISCKAS_spCD28 GYSFTGYFMN WVKQSHGKSL EWIGRIHPYD GDTFYNQNFK DKATLTVDKS _GITR_CD4SNTAHMELLS LTSEDFAVYY CTRYDGSRAM DYWGQGTTVT VSSGGGGSGG 0GGSGGGGSDI ELTQSPASLA VSLGQRAIIS CKASQSVSFA GTSLMHWYHQKPGQQPKLLI YRASNLEAGV PTRFSGSGSK TDFTLNIHPV EEEDAATYYCQQSREYPYTF GGGTKLEIKA AAGSGGSGIL VKQSPMLVAY DNAVNLSCKYSYNLFSREFR ASLHKGLDSA VEVCVVYGNY SQQLQVYSKT GFNCDGKLGNESVTFYLQNL YVNQTDIYFC KIEVMYPPPY LDNEKSNGTI IHVKGKHLCPSPLFPGPSKP FWVLVVVGGV LACYSLLVTV AFIIFWVQLG LHIWQLRSQCMWPRETQLLL EVPPSTEDAR SCQFPEEERG ERSAEEKGRL GDLWVKKVAKKPTNKAPHPK QEPQEINFPD DLPGSNTAAP VQETLHGCQP VTQEDGKESR ISVQERQ 403OSM_MOV19 MGVLLTQRTL LSLVLALLFP SMASMQVQLQ QSGAELVKPG ASVKISCKAS _spCD28GYSFTGYFMN WVKQSHGKSL EWIGRIHPYD GDTFYNQNFK DKATLTVDKS _CD29_SNTAHMELLS LTSEDFAVYY CTRYDGSRAM DYWGQGTTVT VSSGGGGSGG CD40GGSGGGGSDI ELTQSPASLA VSLGQRAIIS CKASQSVSFA GTSLMHWYHQKPGQQPKLLI YRASNLEAGV PTRFSGSGSK TDFTLNIHPV EEEDAATYYCQQSREYPYTF GGGTKLEIKA AAGSGGSGIL VKQSPMLVAY DNAVNLSCKYSYNLFSREFR ASLHKGLDSA VEVCVVYGNY SQQLQVYSKT GFNCDGKLGNESVTFYLQNL YVNQTDIYFC KIEVMYPPPY LDNEKSNGTI IHVKGKHLCPSPLFPGPSKP FWVLVVVGGV LACYSLLVTV AFIIFWVKLL MIIHDRREFAKFEKEKMNAK WDTGENPIYK SAVTTVVNPK YEGKKKVAKK PTNKAPHPKQEPQEINFPDD LPGSNTAAPV QETLHGCQPV TQEDGKESRI SVQERQ 404 OSM_MOV19MGVLLTQRTL LSLVLALLFP SMASMQVOLQ QSGAELVKPG ASVKISCKAS _spCD28GYSFTGYFMN WVKQSHGKSL EWIGRIHPYD GDTFYNQNFK DKATLTVDKS _CD150_CDSNTAHMELLS LTSEDFAVYY CTRYDGSRAM DYWGQGTTVT VSSGGGGSGG 40GGSGGGGSDI ELTQSPASLA VSLGQRAIIS CKASQSVSFA GTSLMHWYHQKPGQQPKLLI YRASNLEAGV PTRFSGSGSK TDFTLNIHPV EEEDAATYYCQQSREYPYTF GGGTKLEIKA AAGSGGSGIL VKQSPMLVAY DNAVNLSCKYSYNLFSREFR ASLHKGLDSA VEVCVVYGNY SQQLQVYSKT GFNCDGKLGNESVTFYLQNL YVNQTDIYFC KIEVMYPPPY LDNEKSNGTI IHVKGKHLCPSPLFPGPSKP FWVLVVVGGV LACYSLLVTV AFIIFWVRRR GKTNHYQTTVEKKSLTIYAQ VQKPGPLQKK LDSFPAQDPC TTIYVAATEP VPESVQETNSITVYASVTLP ESKKVAKKPT NKAPHPKQEP QEINFPDDLP GSNTAAPVQETLHGCQPVTQ EDGKESRISV QERQ 405 OSM_MOV19MGVLLTQRTL LSLVLALLFP SMASMQVQLQ QSGAELVKPG ASVKISCKAS _spCD8_CDGYSFTGYFMN WVKQSHGKSL EWIGRIHPYD GDTFYNQNFK DKATLTVDKS 28_CD40SNTAHMELLS LTSEDFAVYY CTRYDGSRAM DYWGQGTTVT VSSGGGGSGGGGSGGGGSDI ELTQSPASLA VSLGQRAIIS CKASQSVSFA GTSLMHWYHQKPGQQPKLLI YRASNLEAGV PTRFSGSGSK TDFTLNIHPV EEEDAATYYCQQSREYPYTF GGGTKLEIKA AAGSGGSGFV PVFLPAKPTT TPAPRPPTPAPTIASQPLSL RPEACRPAAG GAVHTRGLDF ACDIYIWAPL AGTCGVLLLSLVITLYCNHR NRSKRSRLLH SDYMNMTPRR PGPTRKHYQP YAPPRDFAAYRSKKVAKKPT NKAPHPKQEP QEINFPDDLP GSNTAAPVQE TLHGCQPVTQ EDGKESRISV QERQ406 OSM_MOV19 MGVLLTQRTL LSLVLALLFP SMASMQVQLQ QSGAELVKPG ASVKISCKAS_spCD8_CD GYSFTGYFMN WVKQSHGKSL EWIGRIHPYD GDTFYNQNFK DKATLTVDKS 40SNTAHMELLS LTSEDFAVYY CTRYDGSRAM DYWGQGTTVT VSSGGGGSGGGGSGGGGSDI ELTQSPASLA VSLGQRAIIS CKASQSVSFA GTSLMHWYHQKPGQQPKLLI YRASNLEAGV PTRFSGSGSK TDFTLNIHPV EEEDAATYYCQQSREYPYTF GGGTKLEIKA AAGSGGSGFV PVFLPAKPTT TPAPRPPTPAPTIASQPLSL RPEACRPAAG GAVHTRGLDF ACDIYIWAPL AGTCGVLLLSLVITLYCNHR NKKVAKKPTN KAPHPKQEPQ EINFPDDLPG SNTAAPVQETLHGCQPVTQE DGKESRISVQ ERQ 407 OSM_MOV19MGVLLTQRTL LSLVLALLFP SMASMQVQLQ QSGAELVKPG ASVKISCKAS _spCD8_CDGYSFTGYFMN WVKQSHGKSL EWIGRIHPYD GDTFYNQNFK DKATLTVDKS 137_CD40SNTAHMELLS LTSEDFAVYY CTRYDGSRAM DYWGQGTTVT VSSGGGGSGGGGSGGGGSDI ELTQSPASLA VSLGQRAIIS CKASQSVSFA GTSLMHWYHQKPGQQPKLLI YRASNLEAGV PTRFSGSGSK TDFTLNIHPV EEEDAATYYCQQSREYPYTF GGGTKLEIKA AAGSGGSGFV PVFLPAKPTT TPAPRPPTPAPTIASQPLSL RPEACRPAAG GAVHTRGLDF ACDIYIWAPL AGTCGVLLLSLVITLYCNHR NRFSVVKRGR KKLLYIFKQP FMRPVQTTQE EDGCSCRFPEEEEGGCEKKV AKKPTNKAPH PKQEPQEINF PDDLPGSNTA APVQETLHGCQPVTQEDGKE SRISVQERQ 408 OSM_MOV19MGVLLTQRTL LSLVLALLFP SMASMQVQLQ QSGAELVKPG ASVKISCKAS _spCD8_CDGYSFTGYFMN WVKQSHGKSL EWIGRIHPYD GDTFYNQNFK DKATLTVDKS 134_CD40SNTAHMELLS LTSEDFAVYY CTRYDGSRAM DYWGQGTTVT VSSGGGGSGGGGSGGGGSDI ELTQSPASLA VSLGQRAIIS CKASQSVSFA GTSLMHWYHQKPGQQPKLLI YRASNLEAGV PTRFSGSGSK TDFTLNIHPV EEEDAATYYCQQSREYPYTF GGGTKLEIKA AAGSGGSGFV PVFLPAKPTT TPAPRPPTPAPTIASQPLSL RPEACRPAAG GAVHTRGLDF ACDIYIWAPL AGTCGVLLLSLVITLYCNHR NRRDQRLPPD AHKPPGGGSF RTPIQEEQAD AHSTLAKIKKVAKKPTNKAP HPKQEPQEIN FPDDLPGSNT AAPVQETLHG CQPVTQEDGK SRISVQERQ 409OSM_MOV19 MGVLLTQRTL LSLVLALLFP SMASMQVOLQ QSGAELVKPG ASVKISCKAS_spCD8_CD GYSFTGYFMN WVKQSHGKSL EWIGRIHPYD GDTFYNQNFK DKATLTVDKS 2_CD40SNTAHMELLS LTSEDFAVYY CTRYDGSRAM DYWGQGTTVT VSSGGGGSGGGGSGGGGSDI ELTQSPASLA VSLGQRAIIS CKASQSVSFA GTSLMHWYHQKPGQQPKLLI YRASNLEAGV PTRFSGSGSK TDFTLNIHPV EEEDAATYYCQQSREYPYTF GGGTKLEIKA AAGSGGSGFV PVFLPAKPTT TPAPRPPTPAPTIASQPLSL RPEACRPAAG GAVHTRGLDF ACDIYIWAPL AGTCGVLLLSLVITLYCNHR NKRKKQRSRR NDEELETRAH RVATEERGRK PHQIPASTPQNPATSQHPPP PPGHRSQAPS HRPPPPGHRV QHQPQKRPPA PSGTQVHQQKGPPLPRPRVQ PKPPHGAAEN SLSPSSNKKV AKKPTNKAPH PKQEPQEINFPDDLPGSNTA APVQETLHGC QPVTQEDGKE SRISVQERQ 410 OSM_MOV19MGVLLTQRTL LSLVLALLFP SMASMQVQLQ QSGAELVKPG ASVKISCKAS _spCD8_GIGYSFTGYFMN WVKQSHGKSL EWIGRIHPYD GDTFYNQNFK DKATLTVDKS TR_CD40SNTAHMELLS LTSEDFAVYY CTRYDGSRAM DYWGQGTTVT VSSGGGGSGGGGSGGGGSDI ELTQSPASLA VSLGQRAIIS CKASQSVSFA GTSLMHWYHQKPGQQPKLLI YRASNLEAGV PTRFSGSGSK TDFTLNIHPV EEEDAATYYCQQSREYPYTF GGGTKLEIKA AAGSGGSGFV PVFLPAKPTT TPAPRPPTPAPTIASQPLSL RPEACRPAAG GAVHTRGLDF ACDIYIWAPL AGTCGVLLLSLVITLYCNHR NQLGLHIWQL RSQCMWPRET QLLLEVPPST EDARSCQFPEEERGERSAEE KGRLGDLWVK KVAKKPTNKA PHPKQEPQEI NFPDDLPGSNTAAPVQETLH GCQPVTQEDG KESRISVQER Q 411 OSM_MOV19MGVLLTQRTL LSLVLALLFP SMASMQVQLQ QSGAELVKPG ASVKISCKAS _spCD8GYSFTGYFMN WVKQSHGKSL EWIGRIHPYD GDTFYNQNFK DKATLTVDKS _CD29_SNTAHMELLS LTSEDFAVYY CTRYDGSRAM DYWGQGTTVT VSSGGGGSGG CD40GGSGGGGSDI ELTQSPASLA VSLGQRAIIS CKASQSVSFA GTSLMHWYHQKPGQQPKLLI YRASNLEAGV PTRFSGSGSK TDFTLNIHPV EEEDAATYYCQQSREYPYTF GGGTKLEIKA AAGSGGSGFV PVFLPAKPTT TPAPRPPTPAPTIASQPLSL RPEACRPAAG GAVHTRGLDF ACDIYIWAPL AGTCGVLLLSLVITLYCNHR NKLLMIIHDR REFAKFEKEK MNAKWDTGEN PIYKSAVTTVVNPKYEGKKK VAKKPTNKAP HPKQEPQEIN FPDDLPGSNT AAPVQETLHGCQPVTQEDGK ESRISVQERQ 412 OSM_MOV19MGVLLTQRTL LSLVLALLFP SMASMQVQLQ QSGAELVKPG ASVKISCKAS _spCD8GYSFTGYFMN WVKQSHGKSL EWIGRIHPYD GDTFYNQNFK DKATLTVDKS _CD150_SNTAHMELLS LTSEDFAVYY CTRYDGSRAM DYWGQGTTVT VSSGGGGSGG CD40GGSGGGGSDI ELTQSPASLA VSLGQRAIIS CKASQSVSFA GTSLMHWYHQKPGQQPKLLI YRASNLEAGV PTRFSGSGSK TDFTLNIHPV EEEDAATYYCQQSREYPYTF GGGTKLEIKA AAGSGGSGFV PVFLPAKPTT TPAPRPPTPAPTIASQPLSL RPEACRPAAG GAVHTRGLDF ACDIYIWAPL AGTCGVLLLSLVITLYCNHR NRRRGKTNHY QTTVEKKSLT IYAQVQKPGP LQKKLDSFPAQDPCTTIYVA ATEPVPESVQ ETNSITVYAS VTLPESKKVA KKPTNKAPHPKQEPQEINFP DDLPGSNTAA PVQETLHGCQ PVTQEDGKES RISVQERQ 413 OSM_MOV19MGVLLTQRTL LSLVLALLFP SMASMQVQLQ QSGAELVKPG ASVKISCKAS _spIG4GYSFTGYFMN WVKQSHGKSL EWIGRIHPYD GDTFYNQNFK DKATLTVDKS _CD28_SNTAHMELLS LTSEDFAVYY CTRYDGSRAM DYWGQGTTVT VSSGGGGSGG CD40GGSGGGGSDI ELTQSPASLA VSLGQRAIIS CKASQSVSFA GTSLMHWYHQKPGQQPKLLI YRASNLEAGV PTRFSGSGSK TDFTLNIHPV EEEDAATYYCQQSREYPYTF GGGTKLEIKA AAGSGGSGES KYGPPCPSCP APEFLGGPSVFLFPPKPKDT LMISRTPEVT CVVVDVSQED PEVQFNWYVD GVEVHNAKTKPREEQFNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAKGQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENNYKTTPPVLDS DGSFFLYSRL TVDKSRWQEG NVFSCSVMHE ALHNHYTQKSLSLSLGKMFW VLVVVGGVLA CYSLLVTVAF IIFWVRSKRS RLLHSDYMNMTPRRPGPTRK HYQPYAPPRD FAAYRSKKVA KKPTNKAPHP KQEPQEINFPDDLPGSNTAA PVQETLHGCQ PVTQEDGKES RISVQERQ 414 OSM_MOV19MGVLLTQRTL LSLVLALLFP SMASMQVOLQ QSGAELVKPG ASVKISCKAS _spIG4_GYSFTGYFMN WVKQSHGKSL EWIGRIHPYD GDTFYNQNFK DKATLTVDKS CD40SNTAHMELLS LTSEDFAVYY CTRYDGSRAM DYWGQGTTVT VSSGGGGSGGGGSGGGGSDI ELTQSPASLA VSLGQRAIIS CKASQSVSFA GTSLMHWYHQKPGQQPKLLI YRASNLEAGV PTRFSGSGSK TDFTLNIHPV EEEDAATYYCQQSREYPYTF GGGTKLEIKA AAGSGGSGES KYGPPCPSCP APEFLGGPSVFLFPPKPKDT LMISRTPEVT CVVVDVSQED PEVQFNWYVD GVEVHNAKTKPREEQFNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAKGQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENNYKTTPPVLDS DGSFFLYSRL TVDKSRWQEG NVFSCSVMHE ALHNHYTQKSLSLSLGKMFW VLVVVGGVLA CYSLLVTVAF IIFWVKKVAK KPTNKAPHPKQEPQEINFPD DLPGSNTAAP VQETLHGCQP VTQEDGKESR ISVQERQ 415 OSM_MOV19MGVLLTQRTL LSLVLALLFP SMASMQVQLQ QSGAELVKPG ASVKISCKAS _spIG4GYSFTGYFMN WVKQSHGKSL EWIGRIHPYD GDTFYNQNFK DKATLTVDKS _CD137_SNTAHMELLS LTSEDFAVYY CTRYDGSRAM DYWGQGTTVT VSSGGGGSGG CD40GGSGGGGSDI ELTQSPASLA VSLGQRAIIS CKASQSVSFA GTSLMHWYHQKPGQQPKLLI YRASNLEAGV PTRFSGSGSK TDFTLNIHPV EEEDAATYYCQQSREYPYTF GGGTKLEIKA AAGSGGSGES KYGPPCPSCP APEFLGGPSVFLFPPKPKDT LMISRTPEVT CVVVDVSQED PEVQFNWYVD GVEVHNAKTKPREEQFNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAKGQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENNYKTTPPVLDS DGSFFLYSRL TVDKSRWQEG NVFSCSVMHE ALHNHYTQKSLSLSLGKMFW VLVVVGGVLA CYSLLVTVAF IIFWVKKVAK KPTNKAPHPKQEPQEINFPD DLPGSNTAAP VQETLHGCQP VTQEDGKESR ISVQERQ 416 OSM_MOV19MGVLLTQRTL LSLVLALLFP SMASMQVQLQ QSGAELVKPG ASVKISCKAS _spIG4GYSFTGYFMN WVKQSHGKSL EWIGRIHPYD GDTFYNQNFK DKATLTVDKS _CD134_CDSNTAHMELLS LTSEDFAVYY CTRYDGSRAM DYWGQGTTVT VSSGGGGSGG 40GGSGGGGSDI ELTQSPASLA VSLGQRAIIS CKASQSVSFA GTSLMHWYHQKPGQQPKLLI YRASNLEAGV PTRFSGSGSK TDFTLNIHPV EEEDAATYYCQQSREYPYTF GGGTKLEIKA AAGSGGSGES KYGPPCPSCP APEFLGGPSVFLFPPKPKDT LMISRTPEVT CVVVDVSQED PEVQFNWYVD GVEVHNAKTKPREEQFNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAKGQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENNYKTTPPVLDS DGSFFLYSRL TVDKSRWQEG NVFSCSVMHE ALHNHYTQKSLSLSLGKMFW VLVVVGGVLA CYSLLVTVAF IIFWVRRDQR LPPDAHKPPGGGSFRTPIQE EQADAHSTLA KIKKVAKKPT NKAPHPKQEP QEINFPDDLPGSNTAAPVQE TLHGCQPVTQ EDGKESRISV QERQ 417 OSM_MOV19MGVLLTQRTL LSLVLALLFP SMASMQVQLQ QSGAELVKPG ASVKISCKAS _spIG4GYSFTGYFMN WVKQSHGKSL EWIGRIHPYD GDTFYNQNFK DKATLTVDKS _CD2_CD40SNTAHMELLS LTSEDFAVYY CTRYDGSRAM DYWGQGTTVT VSSGGGGSGGGGSGGGGSDI ELTQSPASLA VSLGQRAIIS CKASQSVSFA GTSLMHWYHQKPGQQPKLLI YRASNLEAGV PTRFSGSGSK TDFTLNIHPV EEEDAATYYCQQSREYPYTF GGGTKLEIKA AAGSGGSGES KYGPPCPSCP APEFLGGPSVFLFPPKPKDT LMISRTPEVT CVVVDVSQED PEVQFNWYVD GVEVHNAKTKPREEQFNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAKGQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENNYKTTPPVLDS DGSFFLYSRL TVDKSRWQEG NVFSCSVMHE ALHNHYTQKSLSLSLGKMFW VLVVVGGVLA CYSLLVTVAF IIFWVKRKKQ RSRRNDEELETRAHRVATEE RGRKPHQIPA STPQNPATSQ HPPPPPGHRS QAPSHRPPPPGHRVQHQPQK RPPAPSGTQV HQQKGPPLPR PRVQPKPPHG AAENSLSPSSNKKVAKKPTN KAPHPKQEPQ EINFPDDLPG SNTAAPVQET LHGCQPVTQE DGKESRISVQ ERQ418 OSM_MOV19 MGVLLTQRTL LSLVLALLFP SMASMQVOLQ QSGAELVKPG ASVKISCKAS_spIG4 GYSFTGYFMN WVKQSHGKSL EWIGRIHPYD GDTFYNQNFK DKATLTVDKS _GITR_SNTAHMELLS LTSEDFAVYY CTRYDGSRAM DYWGQGTTVT VSSGGGGSGG CD40GGSGGGGSDI ELTQSPASLA VSLGQRAIIS CKASQSVSFA GTSLMHWYHQKPGQQPKLLI YRASNLEAGV PTRFSGSGSK TDFTLNIHPV EEEDAATYYCQQSREYPYTF GGGTKLEIKA AAGSGGSGES KYGPPCPSCP APEFLGGPSVFLFPPKPKDT LMISRTPEVT CVVVDVSQED PEVQFNWYVD GVEVHNAKTKPREEQFNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAKGQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENNYKTTPPVLDS DGSFFLYSRL TVDKSRWQEG NVFSCSVMHE ALHNHYTQKSLSLSLGKMFW VLVVVGGVLA CYSLLVTVAF IIFWVQLGLH IWQLRSQCMWPRETQLLLEV PPSTEDARSC QFPEEERGER SAEEKGRLGD LWVKKVAKKPTNKAPHPKQE PQEINFPDDL PGSNTAAPVQ ETLHGCQPVT QEDGKESRIS VQERQ 419OSM_MOV19 MGVLLTQRTL LSLVLALLFP SMASMQVQLQ QSGAELVKPG ASVKISCKAS _spIG4GYSFTGYFMN WVKQSHGKSL EWIGRIHPYD GDTFYNQNFK DKATLTVDKS _CD29_SNTAHMELLS LTSEDFAVYY CTRYDGSRAM DYWGQGTTVT VSSGGGGSGG CD40GGSGGGGSDI ELTQSPASLA VSLGQRAIIS CKASQSVSFA GTSLMHWYHQKPGQQPKLLI YRASNLEAGV PTRFSGSGSK TDFTLNIHPV EEEDAATYYCQQSREYPYTF GGGTKLEIKA AAGSGGSGES KYGPPCPSCP APEFLGGPSVFLFPPKPKDT LMISRTPEVT CVVVDVSQED PEVQFNWYVD GVEVHNAKTKPREEQFNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAKGQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENNYKTTPPVLDS DGSFFLYSRL TVDKSRWQEG NVFSCSVMHE ALHNHYTQKSLSLSLGKMFW VLVVVGGVLA CYSLLVTVAF IIFWVKLLMI IHDRREFAKFEKEKMNAKWD TGENPIYKSA VTTVVNPKYE GKKKVAKKPT NKAPHPKQEPQEINFPDDLP GSNTAAPVQE TLHGCQPVTQ EDGKESRISV QERQ 420 OSM_MOV19MGVLLTQRTL LSLVLALLFP SMASMQVQLQ QSGAELVKPG ASVKISCKAS _spIG4GYSFTGYFMN WVKQSHGKSL EWIGRIHPYD GDTFYNQNFK DKATLTVDKS _CD150_SNTAHMELLS LTSEDFAVYY CTRYDGSRAM DYWGQGTTVT VSSGGGGSGG CD40GGSGGGGSDI ELTQSPASLA VSLGQRAIIS CKASQSVSFA GTSLMHWYHQKPGQQPKLLI YRASNLEAGV PTRFSGSGSK TDFTLNIHPV EEEDAATYYCQQSREYPYTF GGGTKLEIKA AAGSGGSGES KYGPPCPSCP APEFLGGPSVFLFPPKPKDT LMISRTPEVT CVVVDVSQED PEVQFNWYVD GVEVHNAKTKPREEQFNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAKGQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENNYKTTPPVLDS DGSFFLYSRL TVDKSRWQEG NVFSCSVMHE ALHNHYTQKSLSLSLGKMFW VLVVVGGVLA CYSLLVTVAF IIFWVRRRGK TNHYQTTVEKKSLTIYAQVQ KPGPLQKKLD SFPAQDPCTT IYVAATEPVP ESVQETNSITVYASVTLPES KKVAKKPTNK APHPKQEPQE INFPDDLPGS NTAAPVQETLHGCQPVTQED GKESRISVQE RQ 421 OSM_MOV19MGVLLTQRTL LSLVLALLFP SMASMQVQLQ QSGAELVKPG ASVKISCKAS _spIG4GYSFTGYFMN WVKQSHGKSL EWIGRIHPYD GDTFYNQNFK DKATLTVDKS _CD4 0_tanSNTAHMELLS LTSEDFAVYY CTRYDGSRAM DYWGQGTTVT VSSGGGGSGG demGGSGGGGSDI ELTQSPASLA VSLGQRAIIS CKASQSVSFA GTSLMHWYHQKPGQQPKLLI YRASNLEAGV PTRFSGSGSK TDFTLNIHPV EEEDAATYYCQQSREYPYTF GGGTKLEIKA AAGSGGSGES KYGPPCPSCP APEFLGGPSVFLFPPKPKDT LMISRTPEVT CVVVDVSQED PEVQFNWYVD GVEVHNAKTKPREEQFNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAKGQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENNYKTTPPVLDS DGSFFLYSRL TVDKSRWQEG NVFSCSVMHE ALHNHYTQKSLSLSLGKMFW VLVVVGGVLA CYSLLVTVAF IIFWVAKKPT NKAPHPKQEPQEINFPDDLP GSNTAAPVQE TLHGCQPVTQ EDGKESRISV QERQKKVAKKPTNKAPHPKQ EPQEINFPDD LPGSNTAAPV QETLHGCQPV TQEDGKESRI SVQERQKKVA 422OSM_MOV19 MGVLLTQRTL LSLVLALLFP SMASMQVQLQ QSGAELVKPG ASVKISCKAS _spIG4GYSFTGYFMN WVKQSHGKSL EWIGRIHPYD GDTFYNQNFK DKATLTVDKS _CD40_P22SNTAHMELLS LTSEDFAVYY CTRYDGSRAM DYWGQGTTVT VSSGGGGSGG 7AGGSGGGGSDI ELTQSPASLA VSLGQRAIIS CKASQSVSFA GTSLMHWYHQKPGQQPKLLI YRASNLEAGV PTRFSGSGSK TDFTLNIHPV EEEDAATYYCQQSREYPYTF GGGTKLEIKA AAGSGGSGES KYGPPCPSCP APEFLGGPSVFLFPPKPKDT LMISRTPEVT CVVVDVSQED PEVQFNWYVD GVEVHNAKTKPREEQFNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAKGQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENNYKTTPPVLDS DGSFFLYSRL TVDKSRWQEG NVFSCSVMHE ALHNHYTQKSLSLSLGKMFW VLVVVGGVLA CYSLLVTVAF IIFWVKKVAK KPTNKAAHPKQEPQEINFPD DLPGSNTAAP VQETLHGCQP VTQEDGKESR ISVQERQ 423 PDl_PDl_sMQIPQAPWPV VWAVLQLGWR PGWFLDSPDR PWNPPTFSPA LLVVTEGDNA CD28TMTFTCSFSNTS ESFVLNWYRM SPSNQTDKLA AFPEDRSQPG QDCRFRVTQL _CD28_PNGRDFHMSV VRARRNDSGT YLCGAISLAP KAQIKESLRA ELRVTERRAE CD40VPTAHCPSPL FPGPSKPFWV LVVVGGVLAC YSLLVTVAFI IFWVRSKRSR (dimeric)LLHSDYMNMT PRRPGPTRKH YQPYAPPRDF AAYRSKKVAK KPTNKAPHPK CTP188QEPQEINFPD DLPGSNTAAP VQETLHGCQP VTQEDGKESR ISVQERQ 424 PD1_PD1MQIPQAPWPV VWAVLQLGWR PGWRPGWFLD SPDRPWNPPT FSPALLVVTE _sCD28TM_GDNATFTCSF SNTSESFVLN WYRMSPSNQT DKLAAFPEDR SQPGQDCRFR CD40VTQLPNGRDF HMSVVRARRN DSGTYLCGAI SLAPKAQIKE SLRAELRVTERRAEVPTAHC PSPLFPGPSK PFWVLVVVGG VLACYSLLVT VAFIIFWVKKVAKKPTNKAP HPKQEPQEIN FPDDLPGSNT AAPVQETLHG CQPVTQEDGK ESRISVQERQ 425TIGIT_TIG MRWCLLLIWA QGLRQAPLAS GMMTGTIETT GNISAEKGGS IILQCHLSST ITTAQVTQVNWE QQDQLLAICN ADLGWHISPS FKDRVAPGPG LGLTLQSLTV _CD28TMNDTGEYFCIY HTYPDGTYTG RIFLEVLESS VAEHGARFQI PFWVLVVVGG _CD28_VLACYSLLVT VAFIIFWVRS KRSRLLHSDY MNMTPRRPGP TRKHYQPYAP CD40PRDFAAYRSK KVAKKPTNKA PHPKQEPQEI NFPDDLPGSN TAAPVQETLHGCQPVTQEDG KESRISVQER Q 426 TIGIT_TIGMRWCLLLIWA QGLRQAPLAS GMMTGTIETT GNISAEKGGS IILQCHLSST ITTAQVTQVNWE QQDQLLAICN ADLGWHISPS FKDRVAPGPG LGLTLQSLTV _CD28TM_NDTGEYFCIY HTYPDGTYTG RIFLEVLESS VAEHGARFQI PFWVLVVVGG CD40VLACYSLLVT VAFIIFWVKK VAKKPTNKAP HPKQEPQEIN FPDDLPGSNTAAPVQETLHG CQPVTQEDGK ESRISVQERQ 427 OSM_MOV19MGVLLTQRTL LSLVLALLFP SMASMQVQLQ QSGAELVKPG ASVKISCKAS _PD1GYSFTGYFMN WVKQSHGKSL EWIGRIHPYD GDTFYNQNFK DKATLTVDKS _sCD28TM_SNTAHMELLS LTSEDFAVYY CTRYDGSRAM DYWGQGTTVT VSSGGGGSGG CD28GGSGGGGSDI ELTQSPASLA VSLGQRAIIS CKASQSVSFA GTSLMHWYHQ _CD40KPGQQPKLLI YRASNLEAGV PTRFSGSGSK TDFTLNIHPV EEEDAATYYCQQSREYPYTF GGGTKLEIKA AAGSGGSGRP GWFLDSPDRP WNPPTFSPALLVVTEGDNAT FTCSFSNTSE SFVLNWYRMS PSNQTDKLAA FPEDRSQPGQDCRFRVTQLP NGRDFHMSVV RARRNDSGTY LCGAISLAPK AQIKESLRAELRVTERRAEV PTAHCPSPLF PGPSKPFWVL VVVGGVLACY SLLVTVAFIIFWVRSKRSRL LHSDYMNMTP RRPGPTRKHY QPYAPPRDFA AYRSKKVAKKPTNKAPHPKQ EPQEINFPDD LPGSNTAAPV QETLHGCQPV TQEDGKESRI SVQERQ 428OSM_MOV19 MGVLLTQRTL LSLVLALLFP SMASMQVQLQ QSGAELVKPG ASVKISCKAS _PD1GYSFTGYFMN WVKQSHGKSL EWIGRIHPYD GDTFYNQNFK DKATLTVDKS _sCD28TM_SNTAHMELLS LTSEDFAVYY CTRYDGSRAM DYWGQGTTVT VSSGGGGSGG CD40GGSGGGGSDI ELTQSPASLA VSLGQRAIIS CKASQSVSFA GTSLMHWYHQKPGQQPKLLI YRASNLEAGV PTRFSGSGSK TDFTLNIHPV EEEDAATYYCQQSREYPYTF GGGTKLEIKA AAGSGGSGRP GWFLDSPDRP WNPPTFSPALLVVTEGDNAT FTCSFSNTSE SFVLNWYRMS PSNQTDKLAA FPEDRSQPGQDCRFRVTQLP NGRDFHMSVV RARRNDSGTY LCGAISLAPK AQIKESLRAELRVTERRAEV PTAHCPSPLF PGPSKPFWVL VVVGGVLACY SLLVTVAFIIFWVKKVAKKP TNKAPHPKQE PQEINFPDDL PGSNTAAPVQ ETLHGCQPVT QEDGKESRIS VQERQ429 OSM_MOV19 MGVLLTQRTL LSLVLALLFP SMASMQVQLQ QSGAELVKPG ASVKISCKAS_TIGIT GYSFTGYFMN WVKQSHGKSL EWIGRIHPYD GDTFYNQNFK DKATLTVDKS _sCD28TM_SNTAHMELLS LTSEDFAVYY CTRYDGSRAM DYWGQGTTVT VSSGGGGSGG CD28GGSGGGGSDI ELTQSPASLA VSLGQRAIIS CKASQSVSFA GTSLMHWYHQ _CD4 0KPGQQPKLLI YRASNLEAGV PTRFSGSGSK TDFTLNIHPV EEEDAATYYCQQSREYPYTF GGGTKLEIKA AAGSGGSGMM TGTIETTGNI SAEKGGSIILQCHLSSTTAQ VTQVNWEQQD QLLAICNADL GWHISPSFKD RVAPGPGLGLTLQSLTVNDT GEYFCIYHTY PDGTYTGRIF LEVLESSVAE HGARFQIPFWVLVVVGGVLA CYSLLVTVAF IIFWVRSKRS RLLHSDYMNM TPRRPGPTRKHYQPYAPPRD FAAYRSKKVA KKPTNKAPHP KQEPQEINFP DDLPGSNTAAPVQETLHGCQ PVTQEDGKES RISVQERQ 430 OSM_MOV19MGVLLTQRTL LSLVLALLFP SMASMQVQLQ QSGAELVKPG ASVKISCKAS _TIGITGYSFTGYFMN WVKQSHGKSL EWIGRIHPYD GDTFYNQNFK DKATLTVDKS _sCD28TM_SNTAHMELLS LTSEDFAVYY CTRYDGSRAM DYWGQGTTVT VSSGGGGSGG CD40GGSGGGGSDI ELTQSPASLA VSLGQRAIIS CKASQSVSFA GTSLMHWYHQKPGQQPKLLI YRASNLEAGV PTRFSGSGSK TDFTLNIHPV EEEDAATYYCQQSREYPYTF GGGTKLEIKA AAGSGGSGMM TGTIETTGNI SAEKGGSIILQCHLSSTTAQ VTQVNWEQQD QLLAICNADL GWHISPSFKD RVAPGPGLGLTLQSLTVNDT GEYFCIYHTY PDGTYTGRIF LEVLESSVAE HGARFQIPFWVLVVVGGVLA CYSLLVTVAF IIFWVKKVAK KPTNKAPHPK QEPQEINFPDDLPGSNTAAP VQETLHGCQP VTQEDGKESR ISVQERQ 431 Linker GGGGSGGGGS GGGGS 432Truncated NKILVKQSPM LVAYDNAVNL SCKYSYNLFS REFRASLHKG LDSAVEVCVVCytoplasm YGNYSQQLQV YSKTGFNCDG KLGNESVTFY LQNLYVNQTD IYFCKIEVMYic domain PPPYLDNEKS NGTIIHVKGK HLCPSPLFPG PSKPFWVLVV VGGVLACYSL CD28LVTVAFIIFW VRSKR Variant 433 CD28.CD13NKILVKQSPM LVAYDNAVNL SCKYSYNLFS REFRASLHKG LDSAVEVCVV 7 fusionYGNYSQQLQV YSKTGFNCDG KLGNESVTFY LQNLYVNQTD IYFCKIEVMYPPPYLDNEKS NGTIIHVKGK HLCPSPLFPG PSKPFWVLVV VGGVLACYSLLVTVAFIIFW VRSKRSRLLH SDYMNMTPRR PGPTRKHYQP YAPPRDFAAYRSRFSVVKRG RKKLLYIFKQ PFMRPVQTTQ EEDGCSCRFP EEEEGGCE 434 CD28.CD13NKILVKQSPM LVAYDNAVNL SCKYSYNLFS REFRASLHKG LDSAVEVCVV 4 fusionYGNYSQQLQV YSKTGFNCDG KLGNESVTFY LQNLYVNQTD IYFCKIEVMYPPPYLDNEKS NGTIIHVKGK HLCPSPLFPG PSKPFWVLVV VGGVLACYSLLVTVAFIIFW VRSKRSRLLH SDYMNMTPRR PGPTRKHYQP YAPPRDFAAYRSRRDQRLPP DAHKPPGGGS FRTPIQEEQA DAHSTLAKI 435 CD28.CD2NKILVKQSPM LVAYDNAVNL SCKYSYNLFS REFRASLHKG LDSAVEVCVV fusionYGNYSQQLQV YSKTGFNCDG KLGNESVTFY LQNLYVNQTD IYFCKIEVMYPPPYLDNEKS NGTIIHVKGK HLCPSPLFPG PSKPFWVLVV VGGVLACYSLLVTVAFIIFW VRSKRSRLLH SDYMNMTPRR PGPTRKHYQP YAPPRDFAAYRSKRKKQRSR RNDEELETRA HRVATEERGR KPHQIPASTP QNPATSQHPPPPPGHRSQAP SHRPPPPGHR VQHQPQKRPP APSGTQVHQQ KGPPLPRPRVQPKPPHGAAE NSLSPSSN 436 CD28.CD29NKILVKQSPM LVAYDNAVNL SCKYSYNLFS REFRASLHKG LDSAVEVCVV fusionYGNYSQQLQV YSKTGFNCDG KLGNESVTFY LQNLYVNQTD IYFCKIEVMYPPPYLDNEKS NGTIIHVKGK HLCPSPLFPG PSKPFWVLVV VGGVLACYSLLVTVAFIIFW VRSKRSRLLH SDYMNMTPRR PGPTRKHYQP YAPPRDFAAYRSKLLMIIHD RREFAKFEKE KMNAKWDTGE NPIYKSAVTT VVNPKYEGK 437 CD28.GITRNKILVKQSPM LVAYDNAVNL SCKYSYNLFS REFRASLHKG LDSAVEVCVV fusionYGNYSQQLQV YSKTGFNCDG KLGNESVTFY LQNLYVNQTD IYFCKIEVMYPPPYLDNEKS NGTIIHVKGK HLCPSPLFPG PSKPFWVLVV VGGVLACYSLLVTVAFIIFW VRSKRSRLLH SDYMNMTPRR PGPTRKHYQP YAPPRDFAAYRSQLGLHIWQ LRSQCMWPRE TQLLLEVPPS TEDARSCQFP EEERGERSAE EKGRLGDLWV 438CD28.IL2R NKILVKQSPM LVAYDNAVNL SCKYSYNLFS REFRASLHKG LDSAVEVCVVY fusion YGNYSQQLQV YSKTGFNCDG KLGNESVTFY LQNLYVNQTD IYFCKIEVMYPPPYLDNEKS NGTIIHVKGK HLCPSPLFPG PSKPFWVLVV VGGVLACYSLLVTVAFIIFW VRSKRSRLLH SDYMNMTPRR PGPTRKHYQP YAPPRDFAAYRSERTMPRIP TLKNLEDLVT EYHGNFSAWS GVSKGLAESL QPDYSERLCLVSEIPPKGGA LGEGPGASPC NQHSPYWAPP CYTLKPET 439 CD28.CD40NKILVKQSPM LVAYDNAVNL SCKYSYNLFS REFRASLHKG LDSAVEVCVV fusionYGNYSQQLQV YSKTGFNCDG KLGNESVTFY LQNLYVNQTD IYFCKIEVMYPPPYLDNEKS NGTIIHVKGK HLCPSPLFPG PSKPFWVLVV VGGVLACYSLLVTVAFIIFW VRSKRSRLLH SDYMNMTPRR PGPTRKHYQP YAPPRDFAAYRSKKVAKKPT NKAPHPKQEP QEINFPDDLP GSNTAAPVQE TLHGCQPVTQ EDGKESRISV QERQ440 CD28.CD15 NKILVKQSPM LVAYDNAVNL SCKYSYNLFS REFRASLHKG LDSAVEVCVV0 fusion YGNYSQQLQV YSKTGFNCDG KLGNESVTFY LQNLYVNQTD IYFCKIEVMYPPPYLDNEKS NGTIIHVKGK HLCPSPLFPG PSKPFWVLVV VGGVLACYSLLVTVAFIIFW VRSKRSRLLH SDYMNMTPRR PGPTRKHYQP YAPPRDFAAYRSRRRGKTNH YQTTVEKKSL TIYAQVQKPG PLQKKLDSFP AQDPCTTIYVAATEPVPESV QETNSITVYA SVTLPES 441 CD28.CD2.NKILVKQSPM LVAYDNAVNL SCKYSYNLFS REFRASLHKG LDSAVEVCVV CD40YGNYSQQLQV YSKTGFNCDG KLGNESVTFY LQNLYVNQTD IYFCKIEVMY fusionPPPYLDNEKS NGTIIHVKGK HLCPSPLFPG PSKPFWVLVV VGGVLACYSLLVTVAFIIFW VRSKRSRLLH SDYMNMTPRR PGPTRKHYQP YAPPRDFAAYRSKRKKQRSR RNDEELETRA HRVATEERGR KPHQIPASTP QNPATSQHPPPPPGHRSQAP SHRPPPPGHR VQHQPQKRPP APSGTQVHQQ KGPPLPRPRVQPKPPHGAAE NSLSPSSNKK VAKKPTNKAP HPKQEPQEIN FPDDLPGSNTAAPVQETLHG CQPVTQEDGK ESRISVQERQ 442 CD28(IEV)IEVMYPPPYL DNEKSNGTII HVKGKHLCPS PLFPGPSKPF WVLVVVGGVL VariantACYSLLVTVA FIIFWVRSKR SRLLHSDYMN MTPRRPGPTR KHYQPYAPPR DFAAYRS 443 SH3PTNKAPHP motif2 444 SH3 PTNKAPH motif3 445 TRAF2_mot PKQET if4 446TRAF2_mot PVQET if5 447 TRAF2_mot SVQET if 6 448 TRAF6- QEPQEINF Motif2449 HIS tag DYKDDDDK 450 TRAF2/TRA PFSKEECAFRS F3 binding seq sourcehTNFR2 451 TRAF2/TRA AAPVQETLHGC F 3 binding seq source hCD40 452TRAF2/TRA MLSVEEEGKED F 3 binding seq source hCD30 453 TRAF2/TRATIPIQEDYRKP F 3 binding seq source hCD27 454 TRAF2/TRA STPHQEDGKAW F 3binding seq source hLTR 455 TRAF2/TRA TVAVEETIPST F 3 binding seq sourcehATAR 456 TRAF2/TRA RTPIQEEQADA F 3 binding seq source hOX4 0 457TRAF2/TRA TGAAQEEDACS F 3 binding seq source m41BB 458 TRAF2/TRARCPQEEEGGGG F 3 binding seq source m41BB 459 TRAF2/TRA VQTTQEEDGCS F 3binding seq source h41BB 460 TRAF2/TRA RFPEEEEGGCE F 3 binding seqsource h41BB 461 TRAF2/TRA RTPVQESGYPD F 3 binding seq source bLMPI 462TRAF2/TRA RPPVQETGGGG F 3 binding seq source bLMPI 463 TRAF2/TRAHPPVQETGGGG F 3 binding seq source bLMPI 464 TRAF2/TRA HPPVQETGEGG F 3binding seq source bLMPI 465 TRAF2/TRA HPPIQETGNGG F 3 binding seqsource bLMPl 466 TRAF2/TRA ALSSQEAEEVE F 3 binding seq source LAT 467TRAF2/TRA SVPIQCTDKTD F 3 binding seq source hTANK 468 TRAF2/TRAPHPQQATDDSS F 3 binding seq source hLMPl 469 TRAF2/TRA PYPIQATDGGN F 3binding seq source rLMP1 470 TRAF2/TRA PHPIQATDGAN F 3 binding seqsource rLMP1 471 TRAF2/TRA PYPVQASDGGD F 3 binding seq source rLMP1 472TRAF2/ P/S/A/TXQ/EE TRAF 3 binding seq source Minor Consensus 473TRAF2/TRA PVQE F 3 binding seq source Major Consensus 474 CD40KKVAKKPTNKAPHPKQEPQEINFPDDLPGSNTAA--PVQE-- TRAF2/TRA F 3 variant CD40 WT475 CD40 TLHGCQPVTQEDGKESRI--SVQE--RQ TRAF2/TRA F 3 variant CD40/ v41BB476 CD40 KKVAKKPTNKAPHPKQEPQEINFPDDLPGSNTAA--TQEE-- TRAF2/TRA F 3variant CD40/ vTNFR2 477 CD40 TLHGCQPVTQEDGKESRISVQERQ TRAF2/TRA F 3variant CD40/ vATAR 478 CD40 KKVAKKPTNKAPHPKQEPQEINFPDDLPGSNTAA--SKEE--TRAF2/TRA F 3 variant CD40/ vATAR 479 CD40 TLHGCQPVTQEDGKESRISVQERQTRAF2/TRA F 3 variant CD40/ v41BB 480 TRAF6 PQEINF binding seq hCD40 481TRAF6 PQEIDF binding seq hTRANCE-R 482 TRAF6 PPELRF binding seq Mal 483TRAF6 PEEMSW binding seq TRIF 484 TRAF6 PQENSY binding seq IRAK (1) 485TRAF6 PVESDE binding seq IRAK (2) 486 TRAF6 PEESDE binding seq IRAK (3)487 TRAF6 PEETDE binding seq IRAK- 2 (1) 488 TRAF6 PTENGE bindingseq IRAK- 2 (2) 489 TRAF6 PVEDDE binding seq IRAK- 14 490 TRAF6 PPENYEbinding seq RIP2 491 TRAF6 PSELRF binding seq MyD88 492 TRAF6 PXEXXAc/Arbinding seq Consensus 493 CD40KKVAKKPTNKAPHPKQE--PQEINF--PDDLPGSNTAAPVQE TRAF6 variants CD40 WT 494CD40 TLHGCQPVTQEDGKESRISVQERQ TRAF6 variants CD40/ vTRIF 495 CD40KKVAKKPTNKAPHPKQE PEEMSW PDDLPGSNTAAPVQE TRAF6 variants CD40/ vRIP2 496CD40 TLHGCQPVTQEDGKESRISVQERQ TRAF6 variants CD40/ vIRAK(1) 497 CD40KKVAKKPTNKAPHPKQE--PQEINF--PDDLPGSNTAAPVQE TRAF2/TRA F3/ TRAF6 consensusvariants CD40 WT 498 CD40 TLHGCQPVTQEDGKESRISVQERQ TRAF2/TRA F3/ TRAF6consensus variants 499 CD40 KKVAKKPTNKAPHPKQE--PXEXX(Ac/Ar)-- TRAF2/TRAPDDLPGSNTAAPVQETLHGCQPVTQEDGKESRISVQERQ F3/ TRAF6 consensus variants 500CD40 KKVAKKPTNKAPHPKQEPQEINFPDDLPGSNTAA--(P/S/A/T)X(Q/E)E- TRAF2/TRATLHGCQPVTQEDGKESRISVQERQ F3/ TRAF6 consensus variants 501 CD40KKVAKKPTNKAPHPKQEPQEINFPDDLPGSNTAAPVQE TRAF2/TRA F3/ TRAF6 consensusvariants 502 CD40 TLHGCQPVTQEDGKESRI (P/S/A/T)X(Q/E)E-RQ TRAF2/TRAF3/ TRAF6 consensus variants 503 CD40KKVAKKPTNKAPHPKQE--PXEXX(Ac/Ar)--PDDLPGSNTAA-- TRAF2/TRA(P/S/A/T)X(Q/E)-ETLHGCQPVTQEDGKESRISVQERQ F3/ TRAF6 consensus variants504 CD40 KKVAKKPTNKAPHPKQEPQEINFPDDLPGSNTAA--(P/S/A/T)X(Q/E)E- TRAF2/TRATLHGCQPVTQEDGKESRI--(P/S/A/T)X(Q/E)E-RQ F3/ TRAF6 consensus variants 505CD40 KKVAKKPTNKAPHPKQE--PXEXX(Ac/Ar)--PDDLPGSNTAAPVQE TRAF2/TRAF3/ TRAF6 consensus variants 506 CD28(IEV)NKILVKQSPM LVAYDNAVNL SCKYSYNLFS REFRASLHKG LDSAVEVCVV VariantYGNYSQQLQV YSKTGFNCDG KLGNESVTFY LQNLYVNQTD IYFCKIEV 507 SP-TGITMRWCLLLIWA QGLRQAPLAS G 508 SSIQVQLQQSGPE LEKPGASVKL SCKASGYSFT GYTMNWVKQS HGKSLEWIGLITPYNGASSY NQKFRGKATL TVDKSSSTAY MDLLSLTSED SAVYFCARGGYDGRGFDYWG QGTTVTVSSG GGGSGGGGSG GGGSDIELTQ SPAIMSASPGEKVTMTCSAS SSVSYMHWYQ QKSGTSPKRW IYDTSKLASG VPGRFSGSGSGNSYSLTISS VEAEDDATYY CQQWSKHPLT FGAGTKLEIK 509 M5QVQLVQSGAE VEKPGASVKV SCKASGYTFT DYYMHWVRQA PGQGLEWMGW (humaniseINPNSGGTNY AQKFQGRVTM TRDTSISTAY MELSRLRSDD TAVYYCASGW d SSI)DFDYWGQGTL VTVSSGGGGS GGGGSGGGGS DIVMTQSPSS LSASVGDRVTITCRASQSIR YYLSWYQQKP GKAPKLLIYT ASILQNGVPS RFSGSGSGTDFTLTISSLQP EDFATYYCLQ TYTTPDFGPG TKVEIK 510 HN1QVQLVQSGAE VKRPGASVQV SCRASGYSIN TYYMQWVRQA PGAGLEWMGVINPSGVTSYA QKFQGRVTLT NDTSTNTVYM QLNSLTSADT AVYYCARWALWGDFGMDVWG KGTLVTVSSG GGGSGGGGSG GGGSDIQMTQ SPSTLSASIGDRVTITCRAS EGIYHWLAWY QQKPGKAPKL LIYKASSLAS GAPSRFSGSGSGTDFTLTIS SLQPDDFATY YCQQYSNYPL TFGGGTKLEI K 511 M912QVQLQESGPG LVKPSETLSL TCTVSGGSVS SGSYYWSWIR QPPGKGLEWIGYIYYSGSTN YNPSLKSRVT ISVDTSKNQF SLKLSSVTAA DTAVYYCAREGKNGAFDIWG QGTMVTVSSG GGGSGGGGSG GGGSDIQMTQ SPSSLSASVGDRVTITCRAS QSISSYLNWY QQKPGKAPKL LIYAASSLQS GVPSGFSGSGSGTDFTLTIS SLQPEDFATY YCQQSYSTPL TFGGGTKVEI K 512 HuYP218EVQLVESGGG LVQPGGSLRL SCAASGFDLG FYFYACWVRQ APGKGLEWVSCIYTAGSGST YYASWAKGRF TISRDNSKNT LYLQMNSLRA EDTAVYYCARSTANTRSTYY LNLWGQGTLV TVSSGGGGSG GGGSGGGGSD IQMTQSPSSLSASVGDRVTI TCQASQRISS YLSWYQQKPG KVPKLLIYGA STLASGVPSRFSGSGSGTDF TLTISSLQPE DVATYYCQSY AYFDSNNWHA FGGGTKVEI 513 P4QVQLQQSGPG LVTPSQTLSL TCAISGDSVS SNSATWNWIR QSPSRGLEWLGRTYYRSKWY NDYAVSVKSR MSINPDTSKN QFSLQLNSVT PEDTAVYYCARGMMTYYYGM DVWGQGTTVT VSSGGGGSGG GGSGGGGSQP VLTQSSSLSASPGASASLTC TLRSGINVGP YRIYWYQQKP GSPPQYLLNY KSDSDKQQGSGVPSRFSGSK DASANAGVLL ISGLRSEDEA DYYCMIWHSS AAVFGGGTQL TVLS 514OSM_MFE23 MGVLLTQRTL LSLVLALLFP SMASMQVOLQ QSGAELVRSG TSVKLSCTAS _spCD8_GFNIKDSYMH WLRQGPEQGL EWIGWIDPEN GDTEYAPKFQ GKATFTTDTS CD28_CD40SNTAYLQLSS LTSEDTAVYY CNEGTPTGPY YFDYWGQGTT VTVSSGGGGSGGGGSGGGGS ENVLTQSPAI MSASPGEKVT ITCSASSSVS YMHWFQQKPGTSPKLWIYST SNLASGVPAR FSGSGSGTSY SLTISRMEAE DAATYYCQQRSSYPLTFGAG TKLELKRAAA GSGGSGFVPV FLPAKPTTTP APRPPTPAPTIASQPLSLRP EACRPAAGGA VHTRGLDFAC DIYIWAPLAG TCGVLLLSLVITLYCNHRNR SKRSRLLHSD YMNMTPRRPG PTRKHYQPYA PPRDFAAYRSKKVAKKPTNK APHPKQEPQE INFPDDLPGS NTAAPVQETL HGCQPVTQED GKESRISVQE RQ 515Human_ICO GEINGSANYE MFIFHNGGVQ ILCKYPDIVQ QFKMQLLKGG QILCDLTKTKS_Q9Y6W8 GSGNTVSIKS LKFCHSQLSN NSVSFFLYNL DHSHANYYFC NLSIFDPPPFKVTLTGGYLH IYESQLCCQL KFWLPIGCAA FVVVCILGCI LICWLTKKKYSSSVHDPNGE YMFMRAVNTA KKSRLTDVTL 516 CD40 PKQE TRAF2 motif1 517 CD40PVQE TRAF2 motif2 518 CD40 SVQE TRAF2 motif3 519 CD40 QEPQEINFP TRAF6motif 520 CD40 PKA KKPTNKA motif1 521 CD40 PKA SRISVQE motif2 522CD28 EC ILVKQSPMLV AYDNAVNLSC KYSYNLFSRE FRASLHKGLD SAVEVCVVYGNYSQQLQVYS KTGFNCDGKL GNESVTFYLQ NLYVNQTDIY FCKIEVMYPPPYLDNEKSNG TIIHVKGKHL CPSPLFPGPS KP Ar = aromatic residue; Ac = acidicresidue; X = any amino acid

Although the present invention and its advantages have been described indetail, it should be understood that various changes, substitutions andalterations can be made herein without departing from the spirit andscope of the invention as defined in the appended claims.

Various embodiments are also contemplated in the following numberedarrangements:

1. A chimeric costimulatory antigen receptor (CoStAR) which comprises:an extracellular binding domain that binds to carcinoembryonic antigen(CEA), or an extracellular binding domain that binds to mesothelin(MSLN), operatively linked to a transmembrane domain, anda first signaling domain and an intracellular domain of ICOS or asignaling fragment thereof, ora first signaling domain and an intracellular domain of NTRK1 or asignaling fragment thereof, ora first signaling domain and an intracellular domain of DAP10 or asignaling fragment thereof, ora first signaling domain and a CD40 signaling domain or a signalingfragment thereof, ora first signaling domain and one or more of a TRAF2/TRAF3 sequence, aTRAF6 sequence, a TRAF2 sequence, or an IProx sequence.2. The CoStAR of arrangement 1, wherein the first signaling domaincomprises a signaling domain or signaling fragment of CD2, CD9, CD26,CD27, CD28, CD29, CD38, CD40, CD43, CD46, CD49d, CD55, CD73, CD81, CD82,CD99, CD100, CD134 (OX40), CD137 (41BB), CD150 (SLAM), CD270 (HVEM),CD278 (ICOS), CD357 (GITR), or EphB6.3. The CoStAR of arrangement 1, wherein the CoStAR comprises a secondsignaling domain.4. The CoStAR of arrangement 3, wherein the second signaling domaincomprises a signaling domain or signaling fragment of CD2, CD9, CD26,CD27, CD28, CD29, CD38, CD40, CD43, CD46, CD49d, CD55, CD73, CD81, CD82,CD99, CD100, CD134 (OX40), CD137 (41BB), CD150 (SLAM), CD270 (HVEM),CD278 (ICOS), CD357 (GITR), or EphB6.5. The CoStAR of arrangement 1, wherein the CD40 signaling fragmentcomprises an SH3 motif (KPTNKAPH, SEQ ID NO:35), TRAF2 motif (PKQE, SEQID NO:36, PVQE, SEQ ID NO:37, SVQE, SEQ ID NO:38), TRAF6 motif(QEPQEINFP, SEQ ID NO:39), PKA motif (KKPTNKA, SEQ ID NO:40, SRISVQE,SEQ ID NO:41), or a combination thereof, or is a full length CD40intracellular domain.6. The CoStAR of arrangement 2, wherein the first signaling domaincomprises a full length costimulatory domain.7. The CoStAR of arrangement 1, wherein the extracellular binding domainis operatively linked to the transmembrane domain by a linker and/or aspacer.8. The CoStAR of arrangement 7, wherein the linker comprises from about5 to about 20 amino acids.9. The CoStAR of arrangement 7, wherein the linker or spacer comprisesfrom about 10 to about 250 amino acids.10. The CoStAR of arrangement 1, wherein the CoStAR comprises a secondextracellular binding domain.11. The CoStAR of arrangement 10, wherein the second extracellularbinding domain comprises a ligand binding domain from CD8, CD28, orICOS.12. The CoStAR of arrangement 1, wherein the transmembrane domaincomprises a transmembrane domain from CD28, CD8, ICOS, DAP10, or NTRK.13. The CoStAR of arrangement 1, wherein the transmembrane domaincomprises the transmembrane domain sequence of SEQ ID NO:20, SEQ IDNO:21, or SEQ ID NO:22.14. The CoStAR of arrangement 1, wherein the extracellular bindingdomain comprises an scFv, a peptide, an antibody heavy-chain variabledomain, an antibody light-chain variable domain, or a CEA ligand or aMSLN ligand.15. The CoStAR of any one of arrangements 1 to 14, which furthercomprises a CD3ζ signaling domain at the C-terminus.16. The CoStAR of any one of arrangements 1 to 14, which furthercomprises an N-terminal signal peptide.17. The CoStAR of arrangement 16, wherein the N-terminal signal peptidecomprises the signal peptide of oncostatin M (OSM), CD8a, CD2,interleukin-2 (IL-2), granulocyte-macrophage colony stimulating factor(GM-CSF), or human IgGκ.18. A nucleic acid which encodes the CoStAR of any one of arrangements 1to 17.19. A vector which comprises the nucleic acid of arrangement 18.20. A cell which expresses the CoStAR of any one of arrangements 1 to16.21. The cell of arrangement 20, wherein the cell comprises an alpha-betaT cell, gamma-delta T cell, T regulatory cell, TIL, NKT cell or NK cell.22. The cell of arrangement 20, wherein the cell coexpresses a CAR or aTCR.23. A method of making the cell of arrangement 20, which comprises thestep of transducing or transfecting a cell with a vector of arrangement19.24. A method for preparing a population of cells that express a CoStARof any one of arrangements 1 to 16, which comprisesi) detecting expression of the CoStAR on the surface of cellstransfected or transduced with a vector of arrangement 19; andii) selecting cells which are identified as expressing the CoStAR.25. A cell population which is enriched for cell expression a CoStAR ofany one of arrangements 1 to 16.26. A method for treating a disease in a subject, which comprises thestep of administering a cell according to any of arrangements 20 to 22,or a cell population according to arrangement 25 to the subject.

In some embodiments, CoStAR function can be evaluated by co-incubationwith cells expressing the target antigen of the CoStAR scFv. In someembodiments, the cells used the evaluate the CoStAR functioning areOvcar3 cells. In some embodiments, the cells used to evaluate the CoStarfunctioning are Ovcar-OKT-3 cells. In some embodiments, MSLN expressedon the Ovcar3 cells provides signal 2 to the CoStAR expressing cell. Insome embodiments, OKT-3 expressed on the Ovcar3 cells provides signal 1to the CoStAR expressing cell. In some embodiments, both signal 1 andsignal 2 are provided to the CoStAR expressing cells from the Ovcar-OKT3cells. In some embodiments the target cells are K562 cells. In someembodiments, OKT-3 expressed on the K562 cells provides signal 1 to theCoStAR expressing cell. In some embodiments, both signal 1 and signal 2are provided to the CoStAR expressing cells from the K562-OKT3-CEACAM5cells.

In some embodiments, CoStAR transduced cells exhibit higher foldexpansion than nontransduced T cells exposure to signal 1 and signal 2.In some embodiments, CoStAR transduced cells continue to exhibitenhanced expansion after serial restimulation with target cells. In someembodiments, this enhanced expansion can be stimulated for 1 week, for 2weeks, for 3 weeks, for 4 weeks, for 5 weeks, or for 6 weeks posttransduction with the CoStAR construct. In some embodiments, the targetcells are K562-OKT3-CEACAM5 cells.

In some embodiments, a high level of cells positive for CoStARexpression are detected days after transduction. In some embodiments ahigh level of cells positive for CoStAR expression are detected 1 dayafter transduction, 2 days after transduction, 3 days aftertransduction, 4 days after transduction, 5 days after transduction, 6days after transduction, 1 week after transduction, 2 weeks aftertransduction, 3 weeks after transduction, or 4 weeks after transduction.In some embodiments the percentage of cells positive for CoStARexpression is 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 95%, or greater than 95%.

Various embodiments are also contemplated in the following numberedarrangements:

1. A chimeric costimulatory antigen receptor (CoStAR) which comprises:

an extracellular binding domain that binds to carcinoembryonic antigen(CEA), or an extracellular binding domain that binds to mesothelin(MSLN), operatively linked to a transmembrane domain, and

a first signaling domain and an intracellular domain of ICOS or asignaling fragment thereof, or

a first signaling domain and an intracellular domain of NTRK1 or asignaling fragment thereof, or

a first signaling domain and an intracellular domain of DAP10 or asignaling fragment thereof, or

a first signaling domain and a CD40 signaling domain or a signalingfragment thereof, or

a first signaling domain and one or more of a TRAF2/TRAF3 sequence, aTRAF6 sequence, a TRAF2 sequence, or an IProx sequence.

2. The CoStAR of arrangement 1, wherein the first signaling domaincomprises a signaling domain or signaling fragment of CD2, CD9, CD26,CD27, CD28, CD29, CD38, CD40, CD43, CD46, CD49d, CD55, CD73, CD81, CD82,CD99, CD100, CD134 (OX40), CD137 (41BB), CD150 (SLAM), CD270 (HVEM),CD278 (ICOS), CD357 (GITR), or EphB6.

3. The CoStAR of arrangement 1, wherein the CoStAR comprises a secondsignaling domain.

4. The CoStAR of arrangement 3, wherein the second signaling domaincomprises a signaling domain or signaling fragment of CD2, CD9, CD26,CD27, CD28, CD29, CD38, CD40, CD43, CD46, CD49d, CD55, CD73, CD81, CD82,CD99, CD100, CD134 (OX40), CD137 (41BB), CD150 (SLAM), CD270 (HVEM),CD278 (ICOS), CD357 (GITR), or EphB6.

5. The CoStAR of arrangement 1, wherein the CD40 signaling fragmentcomprises an SH3 motif (KPTNKAPH, SEQ ID NO:35), TRAF2 motif (PKQE, SEQID NO:36, PVQE, SEQ ID NO:37, SVQE, SEQ ID NO:38), TRAF6 motif(QEPQEINFP, SEQ ID NO:39), PKA motif (KKPTNKA, SEQ ID NO:40, SRISVQE,SEQ ID NO:41), or a combination thereof, or is a full length CD40intracellular domain.

6. The CoStAR of arrangement 2, wherein the first signaling domaincomprises a full length costimulatory domain.

7. The CoStAR of arrangement 1, wherein the extracellular binding domainis operatively linked to the transmembrane domain by a linker and/or aspacer.

8. The CoStAR of arrangement 7, wherein the linker comprises from about5 to about 20 amino acids.

9. The CoStAR of arrangement 7, wherein the linker or spacer comprisesfrom about 10 to about 250 amino acids.

10. The CoStAR of arrangement 1, wherein the CoStAR comprises a secondextracellular binding domain.

11. The CoStAR of arrangement 10, wherein the second extracellularbinding domain comprises a ligand binding domain from CD8, CD28, orICOS.

12. The CoStAR of arrangement 1, wherein the transmembrane domaincomprises a transmembrane domain from CD28, CD8, ICOS, DAP10, or NTRK.

13. The CoStAR of arrangement 1, wherein the transmembrane domaincomprises the transmembrane domain sequence of SEQ ID NO:20, SEQ IDNO:21, or SEQ ID NO:22.

14. The CoStAR of arrangement 1, wherein the extracellular bindingdomain comprises an scFv, a peptide, an antibody heavy-chain variabledomain, an antibody light-chain variable domain, or a CEA ligand or aMSLN ligand.

15. The CoStAR of any one of arrangements 1 to 14, which furthercomprises a signaling domain at the C-terminus.

16. The CoStAR of any one of arrangements 1 to 14, which furthercomprises an N-terminal signal peptide.

17. The CoStAR of arrangement 16, wherein the N-terminal signal peptidecomprises the signal peptide of oncostatin M (OSM), CD8a, CD2,interleukin-2 (IL-2), granulocyte-macrophage colony stimulating factor(GM-CSF), or human IgGκ.

18. A nucleic acid which encodes the CoStAR of any one of arrangements 1to 17.

19. A vector which comprises the nucleic acid of arrangement 18.

20. A cell which expresses the CoStAR of any one of arrangements 1 to16.

21. The cell of arrangement 20, wherein the cell comprises an alpha-betaT cell, gamma-delta T cell, T regulatory cell, TIL, NKT cell or NK cell.

22. The cell of arrangement 20, wherein the cell coexpresses a CAR or aTCR.

23. A method of making the cell of arrangement 20, which comprises thestep of transducing or transfecting a cell with a vector of arrangement19.

24. A method for preparing a population of cells that express a CoStARof any one of arrangements 1 to 16, which comprises

-   -   i) detecting expression of the CoStAR on the surface of cells        transfected or transduced with a vector of arrangement 19; and    -   ii) selecting cells which are identified as expressing the        CoStAR.

25. A cell population which is enriched for cell expression a CoStAR ofany one of arrangements 1 to 16.

26. A method for treating a disease in a subject, which comprises thestep of administering a cell according to any of arrangements 20 to 22,or a cell population according to arrangement 25 to the subject.

27. A fusion protein, wherein the fusion protein comprises:

-   -   a binding domain specific for CEA linked to;    -   a transmembrane domain that is linked to;    -   an ICOS domain that is linked to;    -   a CD40 signaling domain.

28. A fusion protein, wherein the fusion protein comprises:

-   -   a binding domain specific for MSLN linked to;    -   a transmembrane domain that is linked to;    -   a CD28 domain that is linked to;    -   a CD40 signaling domain.

29. A fusion protein, wherein the fusion protein comprises:

-   -   a first sequence that is at least 70% identical to SEQ ID NO:        12;    -   a second sequence that is a transmembrane domain;    -   a third sequence that is at least 70% identical to SEQ ID NO:        518; and    -   a fourth sequence that is at least 70% identical to SEQ ID NO:        32.

30. A fusion protein, wherein the fusion protein comprises:

-   -   a first sequence that is at least 70% identical to any one of        SEQ ID NO: 186-191;    -   a second sequence that is a transmembrane domain;    -   a third sequence that is at least 70% identical to SEQ ID NO:        25; and    -   a fourth sequence that is at least 70% identical to SEQ ID NO:        32

31. A fusion protein, wherein the fusion protein comprises:

-   -   a HCDR1 that is an HCDR1 in SEQ ID NO: 12;    -   a HCDR2 that is an HCDR2 in SEQ ID NO: 12;    -   a HCDR3 that is an HCDR3 in SEQ ID NO: 12;    -   a LCDR1 that is an LCDR1 in SEQ ID NO: 12;    -   a LCDR2 that is an LCDR2 in SEQ ID NO: 12;    -   a LCDR3 that is an HCDR3 in SEQ ID NO: 12,        -   wherein 1, 2, 3, 4, 5, or 6 of the LCDRs can include 1, 2,            or 3 point mutations;    -   a second sequence that is a transmembrane domain;    -   a third sequence that is at least 70% identical to SEQ ID NO:        515; and    -   a fourth sequence that is at least 70% identical to SEQ ID NO:        32.

32. The fusion protein of arrangement 31, wherein the fusion proteinfurther comprises a signal peptide sequence that is at least 70%identical to SEQ ID NO: 1.

33. The fusion protein of arrangement 31, wherein the fusion proteinfurther comprises a linker sequence that is at least 70% identical toSEQ ID NO: 18.

34. The fusion protein of arrangement 31, wherein the fusion proteinfurther comprises an ICOS sequence that is at least 70% identical to SEQID NO: 515.

35. The fusion protein of arrangement 31, wherein the fusion proteinfurther comprises an CD40 sequence that is at least 70% identical to SEQID NO: 32.

36. A fusion protein, wherein the fusion protein comprises:

-   -   a HCDR1 that is an HCDR1 in SEQ ID NOs: 186-191;    -   a HCDR2 that is an HCDR2 in SEQ ID NOs: 186-191;    -   a HCDR3 that is an HCDR3 in SEQ ID NOs: 186-191;    -   a LCDR1 that is an LCDR1 in SEQ ID NOs: 186-191;    -   a LCDR2 that is an LCDR2 in SEQ ID NOs: 186-191;    -   a LCDR3 that is an HCDR3 in SEQ ID NOs: 186-191        -   wherein 1, 2, 3, 4, 5, or 6 of the LCDRs can include 1, 2,            or 3 point mutations;    -   a second sequence that is a transmembrane domain;    -   a third sequence that is at least 70% identical to SEQ ID NO:        25; and    -   a fourth sequence that is at least 70% identical to SEQ ID NO:        32

37. The fusion protein of arrangement 36, wherein the fusion proteinfurther comprises a signal peptide sequence that is at least 70%identical to SEQ ID NO: 1.

38. The fusion protein of arrangement 36, wherein the fusion proteinfurther comprises a linker sequence that is at least 70% identical toSEQ ID NO: 18.

39. The fusion protein of arrangement 36, wherein the fusion proteinfurther comprises an CD28 TM sequence that is at least 70% identical toSEQ ID NO: 19.

40. The fusion protein of arrangement 36, wherein the fusion proteinfurther comprises an CD28 sequence that is at least 70% identical to SEQID NO: 25.

41. The fusion protein of arrangement 36, wherein the fusion proteinfurther comprises an CD40 sequence that is at least 70% identical to SEQID NO: 32.

42. A method of cell therapy comprising:

-   -   a) identifying a subject, wherein the subject has cancer that        expresses MSLN or CEA; and    -   b) administering any one or more of the CoSTaRs or fusion        proteins in any one of the preceding arrangements.

43. A method of treating a cancer in a subject that expresses MSLN orCEA, the method comprising:

-   -   a) identifying a subject, wherein the subject has cancer that        expresses MSLN or CEA; and    -   b) administering any one or more of the CoSTaRs or fusion        proteins in any one of the preceding arrangements.

The present invention will be further illustrated in the followingExamples which are given for illustration purposes only and are notintended to limit the invention in any way.

EXAMPLES Example 1—Production of T-Cells Expressing CoStAR Materials andMethods

Construct design—The MFE23 CoStAR consists of an MFE23 derived singlechain antibody fragment nucleotide sequence with an oncostatin M1 leadersequence fused to the entire human CD28 nucleic acid sequence. TheCoStAR nucleotide sequence was codon optimised and gene synthesised byGenewiz Inc. The constructs were cloned into pSF.Lenti (Oxford Genetics)via an XbaI and NheI site.

Construct design—The MFE23 CoStAR consists of a CEA-specific MFE23,humanised (hu) MFE23, CEA6, BW431/26 or huT84.66 derived single chainantibody fragment nucleotide sequence with an oncostatin M1, CD8a, CD2,IL-2, GM-CSF or hIgGκ VIII leader sequence. Each CoStAR has an anextracellular spacer domain derived from CD8 or CD28 or truncated CD28and a signalling domain derived from CD28 and CD40. The constructs werecloned into pSF.Lenti (Oxford Genetics) containing an MND promoter, andseparated from a truncated CD34 marker gene via a P2A cleavage sequence.

Lentiviral Production—Lentiviral production was performed using athree-plasmid packaging system (Cell Biolabs, San Diego, USA) by mixing10 μg of each plasmid, plus 10 μg of the pSF.Lenti lentiviral plasmidcontaining the transgene, together in serum free RPMI containing 50 mMCaCl₂). The mixture was added dropwise to a 50% confluent monolayer of293T cells in 75 cm² flasks. The viral supernatants were collected at 48and 72 h post transfection, pooled and concentrated using LentiPaclentiviral supernatant concentration (GeneCopoeia, Rockville, Md., USA)solution according to the manufacturer's instructions. Lentiviralsupernatants were concentrated 10-fold and used to directly infectprimary human T-cells in the presence of 4 g/ml polybrene(Sigma-Aldrich, Dorset, UK). Peripheral blood mononuclear cells wereisolated from normal healthy donors before activation for 24 hours withT-cell activation and expansion beads (Invitrogen) according to themanufacturer's instructions before addition of lentiviral supernatants.

Cell transduction was assessed 96 hours post infection using CEA.hFcprotein and anti-hFc-PE secondary, plus anti-CD34-APC or by anti-CD34-PEantibodies alone. Cells were then expanded further using ×10 donormismatched irradiated PBMC feeders at a 1:20-1:200 ratio in RPMI+10% FCSwith the addition of 1 μg/ml PHA and 200 IU/ml IL-2. After 14 days thecells were stained as previous and stored ready for assay.

Functionality assays were performed by mixing CoStAR positive ornegative cells with wild-type or OKT3 engineered CEA-Positive LoVo orLS174T cells. Briefly, T-cells were mixed with LoVo cells at varyingratios in 96-well plates and IFN7 or IL-2 measured by ELISA. Theremaining cells were incubated with 1:10 dilution of WST-1 reagent(Sigma, UK) for 30 min before absorbance reading at 450 nm. %Cytotoxicity was determined using the followingequation=100−((Experimental reading−T-cells alone)/(tumour alone))×100.

Proliferation assays were performed by first loading T-cells with 10 μMeFluor450 proliferation dye (eBioscience, UK) for 10 min at 37° C. at aconcentration of 1×10⁷ cells/ml before incubating the cells in 5 volumesof cold T-cell media for 5 min on ice. Cells were then washedexcessively to remove unbound dye and added to cocultures containingtumour cells. Cells were removed at 2, 6 and 10 days, 1:200 dilution ofDRAQ7 added and the cells analysed using a MACSQuant cytometer andMACSQuantify software.

Cell counts for proliferation assays were performed by taking cells fromthe wells and staining with anti-CD2 PerCP eFluor710 antibody(eBioscience, UK) for 20 min in the dark, followed by DRAQ7 staining andcounts made using a MACSQuant analyser.

Results

Primary human T-cells were isolated from Buffy coats obtained from theNHSBT. T-cells were isolated by Ficoll-mediated isolation and T-cellnegative isolation kits (StemCell Technologies). The isolated T-cellswere activated with human T-cell activation and expansion beads(Invitrogen, UK). Cells were incubated with concentrated lentiviralparticles and expanded over a number of days. The lentivirus containedthe DNA sequence of the MFE.CoStAR.2A.tCD34 construct (MFE23.scFv fusedto full length human CD28 co-expressed with truncated human CD34 via a2A cleavage sequence). Successfully transduced cells were furtherexpanded using irradiated feeders as outlined in materials and methods.Donor 1 transduction was measured at 22.69% (17.15 CD34+/CoStAR+ plus5.53% CD34-/CoStAR+), donor 2 was measured at 20.73%, and donor 3 at13.34%. Cells were enriched for CoStAR expression using anti-CD34antibodies to obtain T-cell populations greater than 90% CoStARpositive.

To generate a physiologically relevant in vitro model to test the impactof CoStAR on T-cell activity, the non-transduced and transduced cellswere tested against the CEA+ tumour cell lines LoVo and LS174T. Toenable activation of the T-cells in response to the unmatched tumourlines we engineered the tumour cells to express an anti-CD3 single chainantibody fragment anchored to the cell membrane by way of a synthetictransmembrane domain and split from the GFP marker gene using an IRESelement to visualise transduced cells using flow cytometry.

Single cell clones of LoVo and LS174T were generated from bulktransfectants. Non-transduced and CoStAR transduced T-cells were mixedat varying effector:target ratios with wild-type non-transduced orOKT3-engineered LS174T or LoVo cells. After 24 hours coculture media wastaken for IL-2 ELISA measurement. Activation dependent IL-2 secretionwas observed from both CoStAR+ and CoStAR− T-cell populations from threedonors in response to OKT3 engineered LS174T cells with only backgroundIL-2 secretion seen from transduced and non-transduced T-cells inresponse to un-engineered tumour cells (FIG. 3 A-C). CoStAR enhancedIL-2 secretion towards OKT3 engineered tumour cells was found in allthree donors tested. The effect was most evident at E:T ratios of 8:1and 16:1 and at higher E:T ratios IL-2 secretion was too low to measureaccurately. At lower effector to target ratios it appeared that IL-2secretion was saturating from non-transduced cells. These observationswere repeated in LoVo cells with two of the three donors tested againstLS174T with similar results (FIGS. 3D & E).

To determine the impact of CoStAR on T-cell expansion, transduced ornon-transduced T-cells were mixed with wild-type or OKT3-GFP engineeredLoVo cells the number of total cells after 3 days was counted. CoStARenhanced survival and/or proliferation of engineered T-cells in responseto LoVo-OKT3 but not wild-type LoVo cells in the presence of IL-2 (FIG.4A) and absence of IL-2 (FIG. 4B). To further investigate thisphenomenon, cell proliferation analysis was performed in T-cells fromtwo donors using proliferation dye to count the number of cell cycleseach population went through over 6 days (FIGS. 4C & D). A largerproportion of CoStAR engineered cells went through 5, 6 or 7proliferation cycles over 6 days compared to non-engineered cells inresponse to LoVo-OKT3, whereas CoStAR transduced and non-transducedcells went through an average of approximately 2 cycles over the sameduration in response to wild-type LoVo.

A variety of fusion receptors consisting of CD28 fused to an N-terminaladditional costimulatory domain were generated. Costimulatory domainsobtained from: CD137, CD2, CD29, CD134, CD150, CD40, GITR and thesignalling domain from the IL-2 receptor γ-chain (IL2RT) were chosen. Areceptor as close to that used in previous studies of induciblecostimulation was included. This receptor designated CD28(IEV) istruncated such that the C-terminal motif of CD28 is the amino acid triad‘IEV’. Sequences were generated de novo by Genewiz and cloned into alentiviral vector under an EF1α promoter along with a CD34 marker geneseparated from the fusion CoStAR by a 2A self-cleaving peptide. PrimaryCD8+ T-cells were isolated using EasySep beads (StemCell Technologies)and activated with anti-CD3/anti-CD28 activation/expansion Dynabeadsbefore addition of lentiviral particles. Following a short expansionperiod the cells were mixed with LoVo or LoVo-OKT3 cells, with theinclusion of anti-CD107a antibodies and brefeldin and monensin, andfollowing a 16 hour incubation were fixed and stained with antibodies tothe marker gene (CD34) as well as antibodies to IL-2, IFN7 and bcl-xL.Analysis was performed using a MACSQuant analyser and MACSQuantifysoftware. FIG. 5 shows the IL-2 response from CD34− (CoStARnon-transduced) and CD34+(CoStAR transduced). Statistical analysisdemonstrated that all receptors tested induced a significant increase inthe proportion of cells producing IL-2 when harbouring the variantCoStAR receptors. Three other read outs were concurrently measured:IFNγ, a cytokine released under normal signal 1 conditions but enhancedby costimulation; CD107a, a marker of degranulation; and bcl-xL, anantiapoptotic protein upregulated by costimulation. Engagement of CoStARenhanced all the effector functions analysed to varying degrees.CD28.CD2 and CD28.CD40 fusions receptors appeared to elicit the mostrobust response of all the receptors tested (See FIG. 6 )

Example 2

The effect of CD28 and CD28.CD40 based CoStARs on population basedcytokine secretion was compared. Primary T-cells from three donors weretransduced with either the CD28(IEV) truncated CoStAR, full length CD28CoStAR or CD28.CD40 CoStAR (having the full length CD28 as shown in SEQID NO:439, but lacking the N terminal N and K residues) or leftnon-transduced. T-cells were enriched for CoStAR expression using theCD34 marker gene, and following expansion cells were mixed withLoVo-OKT3 cells and IL-2 secretion analysed by ELISA (See FIG. 7 ).Non-transduced cells on average produced 0.80 ng/ml IL-2, with CD28(IEV)and full length CD28 CoStAR producing 4.6 and 5.0 ng/ml IL-2respectively. However CD28.CD40 induced 29.0 ng/ml IL-2 on averageacross three donors thus demonstrating a clear benefit to incorporatingCD40 into the basic CD28-based CoStAR.

Next the effect of CoStAR on T-cell expansion was analysed. T-cells fromseven donors were transduced with either CD28 or CD28.CD40 CoStARs witheither an anti-CA125 (196-14) or anti-Folate receptor (MOV-19) scFv, oran anti-Folate receptor peptide (C7) antigen binding domain. Additionalcells were transduced with a CD28 CoStAR harboring an anti-CEA scFv as amismatched control. Cells were then mixed with CA125+/Folatereceptor+/CEA− cell line OvCAR3 engineered to express a membrane boundOKT3 (OvCAR-OKT3). T-cell counts were made after 7, 14 and 21 days, andfresh OvCAR− OKT3 added at days 7, and 14. Limited expansion of cellsharbouring the anti-CA125 scFv was observed (mean fold expansion: CD28:15.1; CD28.CD40: 69.1), however cells targeting Folate receptor with anscFv did expand in both the CD28 and CD28.CD40 cohorts (mean foldexpansion: CD28: 186.7; CD28.CD40: 1295.0). More limited expansion wasseen when the C7 peptide was used to target the Folate receptor (meanfold expansion: CD28: 71.5; CD28.CD40: 28.0). The control CEA targetingreceptor demonstrated limited expansion (mean fold expansion: 28.0).

To better understand the synergy of signal 1 and signal 2 T-cells wereengineered with a murine constant domain modified TCR which recognizes aCEA peptide (691-699) in the context of HLA-A*02 as well as the CD28 orCD28.CD40 CoStAR targeted towards cell surface CEA protein. As a controlcells were also transduced with a CA125 specific CD28 CoStAR. TheT-cells were mixed with HLA-A*02+/CEA+H508 cells and cytokine productionanalysed by intracellular flow cytometry staining. Flow cytometricgating was performed using antibodies directed towards the murine TCRβconstant domain (marks the TCR engineered cells) as well as the DYKDDDDK(SEQ ID NO:449) epitope tag (marks the CoStAR engineered cells). Thus itwas possible to analyse the TCR−/CoStAR−, TCR+/CoStAR−, TCR−/CoStAR+ andTCR+/CoStAR+ cells in each coculture well. Cytokine production was thenplotted in each subpopulation in either the CD4+ or CD8+ T-cells (FIG. 9). In CD4+ cells CD28.CD40 CoStAR enhanced CD137 and TNFα productionabove TCR stimulation alone, however the TCR response in CD4+ cells waspoor due to the dependency of the TCR on CD8. In CD8+ cells there wasmore robust effector activity with IL-2 and CD107a in particular showinga stronger induction in the CD28.CD40 CoStAR groups. To better comparethe receptors the effector activity in just the TCR+/CoStAR+ groups wasplotted in CD4+ and CD8+ cells (FIG. 10 ). In CD4+ cells induction ofCD137 was significantly enhanced by CD28.CD40 compared to either CEA ormismatched targeting CD28 CoStAR. In CD8+ cells CD137 induction wassignificantly increased compared to either CEA or mismatched targetingCD28 CoStAR, whereas CD107a induction was increased compared to thecontrol CoStAR. Thus CD28.CD40 shows enhanced effector activity across abroad range of models and effector activities.

Example 3

To evaluate costimulation by CD40 bearing CoStARs, primary human T-cellswere mock transduced or transduced with MFE23.CD28 or MFE23.CD28.CD40CoStAR, each harbouring a CD34 marker gene separated by a 2A cleavagepeptide. MFE23 is a single chain Fv antibody that has a high affinityfor carcinoembryonic antigen (CEA). Following in vitro culture cellswere enriched for CD34 using MACS™ paramagnetic selection reagents(Miltenyi Biotech) and then the cells expanded in number usingirradiated feeder cells. MFE23.CD28 CoStAR strongly mediated expansionof CD34⁺ T cells, and MFE23.CD28.CD40 CoStAR further enhanced expansion(FIG. 11 ).

To evaluate costimulatory activity and persistence, T cells mocktransduced or transfected with MFE23.CD28 or MFE23.CD28.CD40 werecocultured with LoVo-OKT3 cells at an 8:1 effector:target ratio in thepresence (200 IU/ml) or absence of exogenous IL-2. At days 1, 4, 7, 11and 18 cells were taken and the number of viable T-cells enumerated byusing anti-CD2 reagents on a MACSQuant flow cytometer. In the absence ofstimulation by tumor and IL-2, cells declined in number as would beexpected (FIG. 12A). In the absence of stimulation but presence of IL-2there was a more apparent survival of the cells, but no specific growth(FIG. 12B). In the presence of tumor, but absence of IL-2 mock cells didnot show specific survival. MFE23.CD28 CoStAR mediated an apparentdoubling in expansion over the first four days followed by decline.MFE23.CD28.CD40 mediated a greater expansion up to day 7 followed by asteady decline (FIG. 12C). Under the same conditions but in the presenceof IL-2 both mock and MFE23.CD28 transduced cells demonstrated a 20-foldexpansion over 18 days, whereas MFE23.CD28.CD40 cells expanded by over60-fold (FIG. 12D). Thus CD28.CD40 based receptors demonstrated superiorexpansion and survival under conditions of stimulation both in thepresence and absence of exogenous IL-2.

Mock transduced and T cells transduced with MFE23.CD28 orMFE23.CD28.CD40 CoStARs were then tested for cytokine production. Beadarray analysis was performed on supernatants obtained from T-cell/tumourcocultures. Engineered T-cells were incubated at a 1:1 effector:targetratio with LoVo-OKT3 cells for 24 hours and supernatant collected.Conditioned supernatant was also collected from an equal number ofT-cells alone, or LoVo-OKT3 cells alone. Production of IL-2, IFN-γ,TNFα, IL-4, IL-5, IL-13, IL-17A, IL-17F, IL-22, IL-6, IL-10, IL-9, andIL-21 was analysed using a Legendplex™ Human TH1/TH2 cytokine panel(Biolegend) (FIG. 13A-13M). Cytokines were either very low orundetectable in media from T-cells or tumour alone. However whencocultured with tumour cytokine production was enhanced. MFE23.CD28enhanced production of IL-2, IL-5, IL-17A/17F, IL-10, IL-9 and IL-21compared to mock. However, MFE23.CD28.CD40 also enhanced production ofTNFα, IL-13 and IL-22. MFE23.CD28.CD40 also enhanced the production of anumber of cytokines greater than that elicited by MFE23.CD28 (IL-2, IL-9and IL-17F), but also reduced the production of some cytokines below thelevels seen with MFE23.CD28 (IL-5 and IL-10). Together this datademonstrates that addition of CD40 to CD28-based Costimulatory receptorsenhances and/or modulates their specific activity with respect tochemokine production.

Mock transduced and T cells transduced with MFE23.CD28 orMFE23.CD28.CD40 CoStARs were further tested for chemokine production.Production of IL-8 (CXCL8), IP-10 (CSCL10), Eotaxin (CCL11), TARC(CCL17), MCP-1 (CCL2), RANTES (CCL5), MIP-1a (CCL3), MIG (CXCL9), ENA-78(CXCL5), MIP-3α (CCL20), GROα (CXCL1), I-TAC (CXCL11), and MEP-10 (CCL4)was analysed using a Legendplex™ Human Pro inflammatory chemokine panel.(FIG. 14A-14M). Chemokines were either very low or undetectable in mediafrom T-cells alone. However when cocultured with tumor, chemokineproduction was enhanced. MFE23.CD28 enhanced production of CXCL5,CXCL10, CXCL11, CCL17 and CCL20 compared to mock. However,MFE23.CD28.CD40 also enhanced production of CCL2, CXCL1 and CXCL9.MFE23.CD28.CD40 also further enhanced the production of certaincytokines to a greater amount than that elicited by MFE23.CD28 (CXCL1,CXCL9, CXCL10, CXCL11, CCL17, CCL2, CXCL9, CCL5 and CCL20), whilereducing the production of some cytokines below the levels seen withMFE23.CD28 (CCL4). Together this data demonstrates that addition of CD40to CD28-based Costimulatory receptors enhances and/or modulates theirspecific activity with respect to chemokine production.

CoStARs were tested for functional activity against cancer targets.Cells were transduced with CD28 or CD28.CD40 CoStARs engineered with anscFv binding domain specific for FolR or CA125 (scFv MOV19 and scFv196-14 respectively). Human folate receptor alpha (FolR) represents asuitable target for a number of tumours including ovarian, head andneck, renal and lung and CA125 represents an alternative target forovarian cancer. Primary human T-cells from six healthy donors wereengineered with either 196-14.CD28, 196-14.CD28.CD40, MOV19.CD28 orMOV19.CD28.CD40 receptors, all harbouring a DYKDDDDK epitope tag fordetection. Transduced cells were mixed with FolR+/CA125+ OvCAR-OKT3cells before analysis of effector activity using intracellular stainingin the epitope tag positive and negative populations. Specificenhancement of effector activity determined by production of IL-2 (FIGS.15A and 15B), TNFα (FIGS. 15C and 15D), CD137 (FIGS. 15E and 15F), andBCL-xL (FIGS. 15G and 15H) was observed in CD28 and CD28.CD40 engineeredcells compared to mock transduce cells in response to both CA125 andFolR, although specific BCL-xL induction by MOV19.CD28 was notsubstantial as compared to MOV19.CD28.CD40.

Mock transduced TILs or TILs engineered with MOV19.CD28.CD40 CoStAR wereevaluated for expansion and CD137 production stimulated by patientmatched tumour digest (FIG. 16 ). Three donor tumours were tested whichdisplayed varying levels of FolR on the digest, ranging from negative(6A), low expression (6B) to high expression (6C). Mock and CoStARnegative TIL in the CoStAR engineered populations of TIL matched for theFolR negative digest demonstrated similar levels of CD137 upregulationfollowing tumour coculture which was not enhanced by the presence ofCoStAR (FIG. 16D). In the TIL exposed to FolR low expressing digestthere was an enhancement in activity in the CoStAR+ cells compared toCoStAR−, with CD137 expression increasing from <10% to >20% (FIG. 16E).In the TIL exposed to FolR high expressing tumour digest there was anincrease in activity from around 20% in the CoStAR− population, up toapproximately 50% in the CoStAR+ population (FIG. 16F).

A FolR targeting CoStAR was examined for enhancement of effectorfunctions. MOV19.CD28.CD40 enhanced CD137 expression from ˜20% to ˜50%(FIG. 17A), TNFα production from 10% to 15% (FIG. 17B) and IL-2production from 2% to 5% (FIG. 17C) in response to FolR+ tumour digest.

CoStAR mediated stimulation by soluble ligand was also examined. T-cellsfrom three healthy donors were engineered with MOV19.CD28 orMOV19.CD28.CD40 CoStAR and activated with either immobilised OKT3,providing stimulation in the absence of FolR, or with OvCAR-OKT3, toprovide TCR and CoStAR activity. Bcl-XL activity was increased frombetween 10 and 20% across the three donors following OKT3 stimulation(FIG. 18A) whereas IL-2 was increased between 0 and 12% (FIG. 18B) andTNFα increased between 0 and 20% (FIG. 18C). The presence of exogenoussoluble FolR did not enhance any of these particular effector functions.In the presence of OvCAR-OKT3 Bcl-XL induction was enhanced by ˜20% inCD28 CoStAR and by ˜35% in CD28.CD40 CoStAR (FIG. 18D), IL-2 inductionwas enhanced by ˜20% in CD28 CoStAR and 30-50% in CD28.CD40 CoStAR (FIG.18E) and TNFα production was enhanced by 20-30% in CD28 CoStAR and25-50% in CD28.CD40 CoStAR (FIG. 18F). Exogenous soluble FolR did nothave an inhibitory effect on any of these effector functions.

Example 4 Materials and Methods

Construct design—The MFE23, MOV19 and 196-14 CoStAR constructs includean MFE23 (CEA specific), MOV19 (Folate receptor a specific) or 196-14(CA125 specific) derived single chain antibody fragment nucleotidesequence with an oncostatin M1 leader sequence fused to a costimulatorydomain. The costimulatory domains contain an extracellular spacer regionand transmembrane domain derived from human CD8 or CD28 and a signallingdomain of either CD28, CD2 or CD137 and/or wild-type or mutant CD40variants. Some CoStARs detailed herein comprise a human PD1extracellular domain fused to CD28 and CD40. Receptors were cloned witha P2A cleavage sequence and a truncated form of human CD34 to permitdetection of transduced cells. The CoStAR nucleotide sequence was codonoptimised and gene synthesised by Genewiz Inc. The constructs werecloned into a third generation lentiviral vector.

Peripheral blood mononuclear cells were isolated from normal healthydonors before activation for 24 hours with T-cell activation andexpansion beads (Invitrogen) according to the manufacturer'sinstructions before addition of lentiviral supernatants.

Cell transduction was assessed 96 hours post infection using CEA.hFcprotein (R&D Systems) and anti-hFc-PE secondary, plus anti-CD34-APC orby anti-CD34-PE antibodies alone. Cells were then expanded further using×10 donor mismatched irradiated PBMC feeders at a 1:20-1:200 ratio inRPMI+10% FCS with the addition of 30 ng/ml OKT3 and 200 IU/ml IL-2.After 14 days the cells were stained as previous and stored ready forassay.

Functionality assays were performed by mixing CoStAR positive ornegative cells with wild-type or OKT3 engineered CEA-Positive LoVocells. Briefly, T-cells were mixed with LoVo cells at varying ratios in96-well plates. For flow analysis cocultures were incubated withBrefeldin and monensin and anti-CD107a antibodies for 16 hours followingwhich cells were stained with Fixable Viability Dye ef450(eBiosciences), fixed with 4% paraformaldehyde and then permeabilisedusing Fix/Perm wash buffer (BD Biosciences). Cells were then stainedwith anti-CD34 or anti DYKDDDDK antibodies to differentiate between theCoStAR+ and CoStAR-populations, anti-IL-2, anti-TNFα and anti-IFN7antibodies (Biolegend). For soluble analyte analysis supernatants werecollected for analysis by ELISA, cytokine bead array (LEGENDPLEX™ HumanTh Cytokine Panel (12-plex)) or chemokine bead array (LEGENDPLEX™ HumanProinflammatory Chemokine Panel (13-plex).

Proliferation assays were performed by mixing T-cells and tumour cellsat an 8:1 effector:target ratio in complete T-cell media (TCM: RPMIsupplemented with 10% FCS, 0.01 M HEPES and 1% Penicillin/streptomycin,50 mM β-mercaptoethanol) in the presence or absence of IL-2. Cell countswere made at indicated time points and fresh tumour cells were added inrestimulation assays at a final E:T of 8:1. Cell counts forproliferation assays were performed by taking cells from the wells andstaining with anti-CD2 PerCP eFluor710 antibody (eBioscience, UK) for 20min in the dark, followed by DRAQ7 staining and counts made using aMACSQuant analyser.

Example 5 CEA Example 5

PD1 fusion receptors: Primary human T-cells isolated from three separatehealthy donors are transduced with the indicated PD1 fusion CoStARreceptors, or an MFE23.CD28.CD40 CoStAR (positive control) or196-14.CD28.CD40 (negative control). PD1 fusion receptors are chosenbased on the format detailed in Ankri et al. J Immunol 2013;191:4121-4129 and Prosser et al. Molecular Immunology 51 (2012) 263-272,but with the addition of CD40 to the signalling domain. For someexperiments, cells are used following Dynabead removal in flowcytometric based assays. To this end transduced T-cells are coculturedwith LoVo-OKT3 (PDL1+, CEA+, CA125-) in the presence of either Nivolumab(anti-PD1) or an isotype matched (IgG4) control antibody, or no antibodyaddition, and then effector function activity (CD137, IFNγ, TNFα andIL-2) is assessed by flow cytometry in the CD34+ (CoStAR+) and CD34−(CoStAR−) cells. PD1.CD28.CD40 fusion receptor positive cells elicitenhanced effector function activity compared to either non-transducedcells or PD1.CD28.CD40 receptor negative cells in response to LoVo-OKT3cells. This effect is also observed in the presence of IgG4 isotypecontrol incubated wells but not Nivolumanb treated wells. Control cellstransduced with MFE23.CD28.CD40, but not 196-15.CD28.CD40 alsodemonstrate enhanced effector activity in response to LoVo-OKT3.

Transduced cells are enriched via the CD34 cell surface marker andincubated with LoVo-OKT3 cells overnight before analysis of cell culturesupernatant via ELISA and cytokine and chemokine bead array.PD1.CD28.CD40 fusion receptor engineered cells produce more IL-2 andCXCL10 compared to non-transduced cells LoVo-OKT3 cells. This effect isalso observed in the presence of IgG4 isotype control antibody but notNivolumab (anti-PD1). Control cells transduced with MFE23.CD28.CD40, butnot 196-15.CD28.CD40 also demonstrate enhanced IL-2 and CXCL10production in response to LoVo-OKT3.

Signaling domain analysis: Primary human T-cells isolated from threeseparate healthy donors are transduced with the indicated signalingdomain variant CoStARs (MFE23.CD28, MFE23.CD28.CD40,MFE23.CD8ec/tm.CD28.CD40, MFE23.CD8ec/tm.CD40,MFE23.CD8ec/tm.CD137.CD40, MFE23.CD8ec/tm.CD137,MFE23.CD8ec/tm.CD2.CD40, MFE23.CD8ec/tm.CD2), or a 196-14.CD28.CD40(negative control) CoStAR. CD8ec/tm indicates a component comprising aspacer and transmembrane domain from CD8 (e.g., SEQ ID NO:20).Transduced cells are enriched via the CD34 cell surface marker andincubated with LoVo-OKT3 cells overnight before analysis of cell culturesupernatant via ELISA. MFE23.CD28.CD40 produces more IL-2 thanMFE23.CD28, MFE23.CD8ec/tm.CD28.CD40 is very similar in responsivenessto MFE23.CD28.CD40 demonstrating that the choice of spacer domain doesnot impact on CoStAR activity. The addition of CD40 toMFE23.CD8ec/tm.CD137 or MFE23.CD8ec/tm.CD2 enhances the specificactivity of these receptors with regards to IL-2 production andproduction of other cytokines and chemokines, thus demonstrating thataddition of CD40 improves the activity of CoStAR based on receptorsignalling domains other than CD28.

To determine which receptor signaling motifs contribute to CD40enhancement of CoStAR activity, variant receptors are generatedharbouring mutations in the TRAF6 binding motif (MFE23.CD28.CD40 (PQEINFmutated to AQAINF)), the TRAF2 binding motif (MFE23.CD28.CD40 (SVQEmutated to AVQA)) or the TRAF1/2/3/5 binding motif (MFE23.CD28.CD40(PVQET mutated to AVAEA)). Also generated is a receptor with atriplicated CD40 signalling domain MFE23.CD28.CD40(x3), and a receptorwith a P227A mutation. Primary human T-cells isolated from threeseparate healthy donors are transduced with the indicated CoStARs andcells enriched for CoStAR expression via CD34. Cocultures are set upwith LoVo-OKT3 cells and supernatants collected for ELISA andcytokine/chemokine bead array. All receptors harbouring a mutation inany one of the three signalling motifs demonstrate reduced effectoractivity in response to LoVo-OKT3 cells (IL-2 and/or CXCL10). Cellsexpressing the CD28.CD40 (X3) receptor or P227A mutation demonstrateenhanced activity in response to LoVo-OKT3.

To determine which receptor signaling motifs contribute to CD40enhancement of CoStAR activity, variant receptors were generatedharbouring mutations in the TRAF6 binding motif (MFE23.CD28.CD40 (PQEINFmutated to AQAINF)), the TRAF2 binding motif (MFE23.CD28.CD40 (SVQEmutated to AVQA)) or the TRAF1/2/3/5 binding motif (MFE23.CD28.CD40(PVQET mutated to AVAEA)). Primary human T-cells isolated from threeseparate healthy donors were transduced with the indicated CoStARs andcells enriched for CoStAR expression via CD34. Cocultures were set upwith LoVo-OKT3 cells and supernatants collected for cytokine analysis.

IL2 production by non-transduced cells was very low, but elevated incells expressing the wild-type MFE23.CD28.CD40 CoStAR. IL2 productionwas not largely affected by mutations to the SVQE, PQEINF or Q263Amutation. However, mutation to the PVQET motif had a dramatic effect onIL2 production by T-cells.

Concurrently proliferation of engineered T-cells was assessed inresponse to LoVo-OKT3 cells over 2 rounds of stimulation with LoVo-OKT3cells. Non-transduced cells did not survive even one round ofstimulation, whereas cells expressing the wild-type receptor underwent a10-fold expansion over 2 rounds of stimulation over 14-days. Cellsharboring either a SVQE-AVQA mutation, or PQEINF-AQAINF mutationdisplayed a reduced capacity to undergo proliferation, this was furtherexacerbated in cells harboring a Q263A mutation. However, cellsharboring a PVQET-AVAEA mutation in the TRAF2/3 binding region displayeda profound inability to proliferate over 2 rounds of stimulation.

Example 6

To evaluate the in vivo anti-tumor activity of T cells transduced withCD40 bearing CoStARs, primary human T-cells are mock transduced ortransduced with MOV19.CD28.CD40 CoStAR construct followed by in vitroexpansion and cryopreservation. MOV19 is a single chain Fv antibody thathas a high affinity for Folate Receptor alpha (FolR1). Immunocompromisedmice are implanted with an established ovarian cancer cell line (A2870,OVCAR-5, OVCAR-8 or SK-OV-3), which is allowed to grow in the animal forfew days. Mice are subsequently staged according to their tumor burden,and finally injected with the mock transduced T cells or MOV19.CD28.CD40transduced T cells. Shortly after the T cell dosing, some of the miceare injected with intravenous IL-2 (5 g IL-2, Q2Dx7) to support theengraftment and initial expansion of T cells. The final study designcontains 5 groups (each one containing 5 mice): PBS (no cells dosed),mock transduced T cells, mock transduced T cells with IL-2supplementation, MOV19.CD28.CD40 transduced T cells and MOV19.CD28.CD40transduced T cells with IL-2 supplementation. Tumor growth and micesurvival is monitored on weekly basis for a total of 40 days.

Mice administered with MOV19.CD28.CD40 transduced cells show bettertumor control and prolonged survival compared to the mock transducedgroups, whether or not supplemented with IL-2. This data demonstratesthe ability of the CoStAR platform to improve in vivo the T cellanti-tumor response and also illustrates how this improved response isindependent of the presence of exogenous IL-2.

Example 7

Design of CoStARs that bind to CEA—The CoStAR consists of a CEA specificMFE23, humanised (hu) MFE23, CEA6, BW431/26 or huT84.66 derived singlechain antibody fragment nucleotide sequence with an oncostatin M1, CD8a,CD2, IL-2, GM-CSF or hIgGκ VIII leader sequence. Each CoStAR has anextracellular spacer domain derived from CD8 or CD28 or truncated CD28and a signalling domain derived from CD28 and CD40. The constructs arecloned into pSF.Lenti (Oxford Genetics) containing an MND promoter, andseparated from a truncated CD34 marker gene via a P2A cleavage sequence.

Design of CoStARs that bind to MSLN—The CoStAR consists of a MSLNspecific SS1, humanized SS1 (M5), HN1, M912, HuYP218 or P4 derivedsingle chain antibody fragment nucleotide sequence with an oncostatinM1, CD8a, CD2, IL-2, GM-CSF or hIgGκ VIII leader sequence. Each CoStARhas an extracellular spacer domain derived from CD8 or CD28 or truncatedCD28 and a signalling domain derived from CD28 and CD40. The constructswere cloned into pSF.Lenti (Oxford Genetics) containing an MND promoter,and separated from a truncated CD34 marker gene via a P2A cleavagesequence.

Lentiviral Production—Lentiviral production is performed using athree-plasmid packaging system (Cell Biolabs, San Diego, USA) by mixing10 μg of each plasmid, plus 10 μg of the pSF.Lenti lentiviral plasmidcontaining the transgene, together in serum free RPMI containing 50 mMCaCl₂). The mixture is added dropwise to a 50% confluent monolayer of293T cells in 75 cm2 flasks. The viral supernatants are collected at 48and 72 h post transfection, pooled and concentrated using Lenti-Xlentiviral supernatant concentration (Takara Bio Inc. Japan) solutionaccording to the manufacturer's instructions. Lentiviral supernatantsare concentrated 10-fold and used to directly infect primary humanT-cells at an MOI of 3-5 in the presence of 4 μg/ml polybrene(Sigma-Aldrich, Dorset, UK). Peripheral blood mononuclear cells areisolated from normal healthy donors before activation for 24 hours withT-cell activation and expansion beads (Invitrogen) according to themanufacturer's instructions before addition of lentiviral supernatants.

Cell transduction is assessed 96 hours post infection using CEA.hFcprotein and anti-hFc-PE secondary, plus anti-CD34-APC or by anti-CD34-PEantibodies alone. Cells are then expanded further using ×10 donormismatched irradiated PBMC feeders at a 1:200 ratio in T-cell media(RPMI 1640, 10% FBS, 10 mM HEPES, 50 μM β-mercaptoethanol and 50 u/mlPenicillin/streptomycin), 200 IU/ml IL-2 and a final concentration of 30ng/ml anti-CD3 (OKT3). After 12-14 days the cells are stained asprevious and stored ready for assay.

Functionality assays are performed by mixing CoStAR positive or negativecells with wild-type or OKT3 engineered CEA or MSLN positive cell lines(LoVo, HT29, SW480, H508). Briefly, T-cells are mixed with target cellsat varying ratios in 96-well plates and cytokine release measured byELISA and MSD analysis.

Cytotoxicity assays are performed using the xCELLigence RTCA SP realtime cell analyser system (Acea). A programme was generated to test wellconductivity (cell index) of a 96-well PET E-plate every 15 min for theduration of the experiment (up to 250 hr). 50 μL T cell medium was addedto the wells which were to have cell index tested and incubated at roomtemperature (RT) for 30 minutes. The E-plate was added to the RTCA SPdevice and background conductivity readings measured.

The optimal density of target cells to seed is defined as that whichreaches a stable cell index between 24- and 36-hours after the beginningof the assay and does not decrease without intervention (i.e. additionof Triton-x-100 or effector cell populations) before the end of theassay. Target cells at optimal density for killing assays (cell linedependent) are counted using a quantitative method capable of dead-celldiscrimination. The E-plate is removed from the device and the optimaldensity of live cells is added to the wells containing T cell medium ata final volume of 100 μL before incubation for 30 minutes at RT.

The E-plate us then placed back on the analyser and cell index valuesacquired until a stable cell index is observed, at which point theprogramme is paused and the E-plate removed from the RTCA SP device.Treatments are added to the appropriate wells. Treatments consist ofeither 100 μL T cell medium (no treatment control), or the same volumecontaining effector cells or 0.5% Triton-x-100 (full lysis control).Effector cell counting uses a quantitative method capable of dead- andapoptotic-cell discrimination. The number of effectors to target cellsvaried depending on the experiment.

During data analysis, the cell index is normalised using the followingequation in the RTCA software package:

NormalisedCIti=CIti/CInml_time

Where CIti=Cell index (CI) at a specific time point, and CInml_time=CIat the time point prior to addition of T cells.

Data is then further manipulated relative to the full lysis control togive % cytolysis:

% Cytolysisst=[1−(NCIst)/(AvgNCIRt)]×100

where, NCIst is the Normalised Cell Index for the sample and NCIRt isthe average of Normalise Cell Index for the matching reference wells.

Repeat stimulation assays are performed by mixing 5×10⁴ CoStARtransduced or mock-transduced cells at an 8:1 E:T ration with LoVo-OKT3cells in the absence of exogenous IL-2, in triplicate well of a 96-wellU-bottom plate. T-cell counts are made on D1, 4 and 7 via flowcytometric assessment of numbers based on αCD2 gating, and fresh tumourcells added at seven day intervals. Relative expansion is assessed bysplitting of wells and enumeration of fold change based upon theoriginal seeding density with the proportion of cells removed forcounting also factored in.

Results

A model system is developed to evaluate the impact of various structuralcomponents of CEA and MSLN directed CoStAR receptors. To this end thesignal peptide (SP), single chain antibody fragment (scFv) andextracellular spacer (ES) are assessed for their impact on expressionand function.

The impact of the signal peptide on expression of the CoStAR is tested,as it is known that different signal peptides can affect expression ofvarious recombinant proteins (REF). The MFE23.CD28.CD40 CoStAR receptorsare generated with various different leader sequences (which encode thedesired signal peptide) sequences. These include signal peptides derivedfrom: Oncostatin M1 (OSM), IL2, CD2, CD8a, GMCSF and hIgGκ VIII. Eachleader sequence is cloned in frame with the MFE23 scFv sequence togenerate SP.MFE23.CD28.CD40.P2A.tCD34. A Jurkat cell line model isselected to investigate the relative expression of each SP modifiedCoStAR relative to the tCD34 marker gene. To this end Jurkat JRT3-T3.5T-cells are incubated with lentiviral particles at an MOI of 5. Sevendays post transduction the cells are stained with anti-CD34 antibodiesto stain for the transduced cells, and CEA.hFc protein followed byanti-hFc secondary antibodies to identify for the CEA CoStAR. All SPmodified CoStAR variants tested are found to be expressed in the CD34+proportion of the JRT3-T3.5 cells.

Next, the impact of different CEA specific scFv in the context of CoStARis assessed, as well as investigating different spacer domains. Sixdifferent scFvs are compared; as well as the MFE23 sequence describedabove (Chester et al. 1994), including an MFE23 K>Q mutant, humanised(Hu) MFE23 (Begent et al. 2003), CEA6 (Jackson et al. 1998), BW431/26(Seenmann et al. 1991) and HuT84.66 (Yazaki et al. 2005).

Primary human T-cells are isolated from Buffy coats obtained from theNHSBT. T-cells are isolated by Ficoll-mediated isolation and T-cellnegative isolation kits (StemCell Technologies). The isolated T-cellsare activated with human T-cell activation and expansion beads(Invitrogen, UK). Cells are incubated with concentrated lentiviralparticles, encoding CEA CoStARs containing the OSM SP, and expanded overa number of days. Cells are enriched for CoStAR expression usinganti-CD34 antibodies to obtain T-cell populations greater than 90%CoStAR positive before being placed in a rapid expansion protocol (REP),with irradiated buffy coat derived PBMCs as outlined in the materialsand methods.

A physiologically relevant in vitro model is employed to test the impactof CoStAR on T-cell activity. Transduced and non-transduced cells aretested against the CEA+ cell lines LoVo, H508, SW480 or HT29. The murineCEA− cell line Ba/F3 is engineered to express CEA as a control. Toenable activation of the T-cells in response to the unmatched tumourlines the tumour cells are engineered to express an anti-CD3 singlechain antibody fragment anchored to the cell membrane by way of asynthetic transmembrane domain and split from a GFP marker gene using anIRES element to visualise transduced cells using flow cytometry. Celllines are also engineered to express firefly-luciferase (ffLuc) underpuromycin selection to permit analysis of target cell lysis.

Non-transduced and CoStAR transduced T-cells are mixed at varyingeffector:target ratios with wild-type or OKT3-engineered tumour celllines. After 24 hours coculture media is taken for IL-2 ELISAmeasurement. Activation dependent IL-2 secretion is observed from bothCoStAR+ and CoStAR− T-cell populations from all donors in response toOKT3 engineered cells with only background IL-2 secretion seen fromtransduced and non-transduced T-cells in response to un-engineeredtumour cells. In all donors tested, the presence of CEA CoStAR enhanceseffector activity (IL2, IL3, CXCL10) towards OKT3 engineered CEA+ tumourlines.

Cocultures with Ba/F3 cells demonstrate the targeted approach of the CEACoStARs. Coculture of CEA CoStAR engineered cells with Ba/F3 orBa/F3-CEA does not result in specific IL2 release whereas incubationwith Ba/F3-OKT3 enhances IL-2 secretion. However, incubation of T-cellswith Ba/F3-OKT3/CEA significantly enhances IL2 secretion compared toBa/F3-OKT3 alone.

The impact of CoStAR on tumour cell killing is determined. Transduced ornon-transduced T-cells are mixed with wild-type or OKT3-GFP engineeredtumour cells and quantified residual tumour cell derived luciferaseactivity at defined time points. The presence of CoStAR enhances theability of T-cells to mediate target cell lysis. This enhanced abilityof CoStAR+ cells to mediate anti-tumour activity is also evident usingthe xCELLigence device as outlined in materials and methods.

Repeat stimulation assays are performed according to materials andmethods. In brief mock or CoStAR engineered cells are mixed at an 8:1E:T ratio with OKT3 engineered target lines and the relative expansionof T-cells enumerated at the indicated time points, with fresh tumouradded at seven day intervals. All CoStARs tested mediate prolongedsurvival and expansion of T-cells across multiple rounds of stimulation,whereas mock transduced cells decline in number following repeatstimulations.

To evaluate CoStAR activity in TIL specimens, TIL are engineered withCEA specific CoStAR constructs. To this end tumours are digested andanalysed for CEA expression using flow cytometry. Tumour digests testingpositive are engineered with CEA CoStARs. Following outgrowth and rapidexpansion protocol, engineered and matched non-engineered TIL are mixedwith either tumour digest, or where available, matched autologous tumourlines. In all donors tested the presence of the CEA CoStAR enhancesspecific effector activity as measured by IFNγ and IL-2 compared tocells which are mock transduced.

The impact of different MSLN specific scFv in the context of CoStAR isassessed, as well as investigating different spacer domains. Sixdifferent scFvs are compared: SS1 (Chowdhury, 1999), humanised (Hu)-SS1(Begent et al. 2003, M5) (patent CA2931684A1), HN1 (Jackson et al.1998), BW431/26 (Seenmann et al. 1991) and HuT84.66 (Yazaki et al.2005Ho, 2011), M912 (Feng, 2009), HuYP218 (Zhang, 2015) and P4 (patentU.S. Pat. No. 9,272,002B2).

Primary human T-cells are isolated from Buffy coats obtained from theNHSBT. T-cells are isolated by Ficoll-mediated isolation and T-cellnegative isolation kits (StemCell Technologies). The isolated T-cellsare activated with human T-cell activation and expansion beads(Invitrogen, UK). Cells are incubated with concentrated lentiviralparticles, encoding MSLN CoStARs containing the OSM SP, and expandedover a number of days. Cells are enriched for CoStAR expression usinganti-CD34 antibodies to obtain T-cell populations greater than 90%CoStAR positive before being placed in a rapid expansion protocol (REP),with irradiated buffy coat derived PBMCs as outlined in the materialsand methods.

A physiologically relevant in vitro model is employed to test the impactof CoStAR on T-cell activity. Transduced and non-transduced cells aretested against the MSLN+/− cell lines LoVo (MSLN−), H508 MSLN+, SW480(MSLN+) or HT29 (MSLN+). The murine MSLN− cell line Ba/F3 is engineeredto express MSLN as a control. To enable activation of the T-cells inresponse to the unmatched tumour lines the tumour cells are engineeredto express an anti-CD3 single chain antibody fragment anchored to thecell membrane by way of a synthetic transmembrane domain and split froma GFP marker gene using an IRES element to visualise transduced cellsusing flow cytometry. Cell lines are also engineered to expressfirefly-luciferase (ffLuc) under puromycin selection to permit analysisof target cell lysis.

Non-transduced and CoStAR transduced T-cells are mixed at varyingeffector:target ratios with wild-type or OKT3-engineered tumour celllines. After 24 hours coculture media is taken for IL-2 ELISAmeasurement. Activation dependent IL-2 secretion is observed from bothCoStAR+ and CoStAR− T-cell populations from all donors in response toOKT3 engineered cells with only background IL-2 secretion seen fromtransduced and non-transduced T-cells in response to un-engineeredtumour cells. In all donors tested, the presence of MSLN CoStAR enhanceseffector activity (IL2, IL3, CXCL10) towards OKT3 engineered MSLN+tumour lines.

Cocultures with Ba/F3 cells demonstrate the targeted approach of theMSLN CoStARs. Coculture of MSLN CoStAR engineered cells with Ba/F3 orBa/F3-MSLN does not result in specific IL2 release whereas incubationwith Ba/F3-OKT3 enhances IL-2 secretion. However, incubation of T-cellswith Ba/F3-OKT3/MSLN significantly enhances IL2 secretion compared toBa/F3-OKT3 alone.

The impact of CoStAR on tumour cell killing is determined. Transduced ornon-transduced T-cells are mixed with wild-type or OKT3-GFP engineeredtumour cells and quantified residual tumour cell derived luciferaseactivity at defined time points. The presence of CoStAR enhances theability of T-cells to mediate target cell lysis. This enhanced abilityof CoStAR+ cells to mediate anti-tumour activity is also evident usingthe xCELLigence device as outlined in materials and methods.

Repeat stimulation assays are performed according to materials andmethods. In brief mock or CoStAR engineered cells are mixed at an 8:1E:T ratio with OKT3 engineered target lines and the relative expansionof T-cells enumerated at the indicated time points, with fresh tumouradded at seven day intervals. All CoStARs tested mediate prolongedsurvival and expansion of T-cells across multiple rounds of stimulation,whereas mock transduced cells decline in number following repeatstimulations.

To evaluate CoStAR activity in TIL specimens, TIL are engineered withMSLN specific CoStAR constructs. To this end tumours are digested andanalysed for MSLN expression using flow cytometry. Tumour digeststesting positive are engineered with MSLN CoStARs. Following outgrowthand rapid expansion protocol, engineered and matched non-engineered TILare mixed with either tumour digest, or where available, matchedautologous tumour lines. In all donors tested the presence of therelevant CoStAR enhances specific effector activity as measured by IFN7and IL-2 compared to cells which are mock transduced.

Example 8 Anti-MSLN CoStAR Expression

Anti-MSLN CoStARs comprising different scFv antigen-binding domains(Table 8) were compared for surface expression on T cells from healthydonors.

TABLE 8 Clone CTP224 CTP225 CTP226 CTP227 CTP228 CTP229 SEQ ID NO: 192210 228 246 264 282 Signal peptide OSM1 scFv SS1 M5 HN1 M912 huYP218 P4Linker AAAGSGGSG Spacer CD28 EC TM CD28 Intracellular CD28.CD40

T cells from healthy donor (HD) PBMCs were lentivirus transduced, at amultiplicity of infection (MOI) 5, with six variable scFV constructsagainst mesothelin (MSNL) that possessed CD28.CD40 signaling domains.Non-transduced (MOCK) cells were used as controls. Cells were sortedusing CD34 microbeads and underwent a rapid expansion protocol (REP) for14 days. Following expansion, 1×10⁵ cells were assessed for transductionefficiency either via surface detection of the marker gene tCD34 orCoStAR molecule using an anti-CD34-APC (black) or anti-MSLN-PE (red)antibody, respectively. (FIG. 19 ). The results represent 3 biologicalreplicates.

Example 9 Anti-MSLN CoStAR Activity

Cytokine production was assessed in CoStAR transduced HD T cellscocultured with target cell lines. A variety of CoStARs comprisingdifferent anti-MSLN binding domains, spacers, or transmembrane domains(Table 8, Table 9, Table 10) were tested.

TABLE 9 Clone CTP248 CTP249 CTP250 CTP251 CTP252 CTP253 SEQ ID NO: 300306 312 318 324 330 Signal peptide OSM1 scFv SS1 M5 HN1 M912 huYP218 P4Linker AAAGSGGSG Spacer Truncated CD28 EC TM CD28 IntracellularCD28.CD40

TABLE 10 Clone CTP236 CTP237 CTP238 CTP239 CTP240 CTP241 SEQ ID NO: 198216 234 252 270 288 Signal peptide OSM1 scFv SS1 M5 HN1 M912 huYP218 P4Linker AAAGSGGSG Spacer CD8 EC TM CD8 Intracellular CD28.CD40

Nontransduced (MOCK) and anti-MSNL CoStAR transduced HD T cells werecocultured with engineered OVCAR3 target cell lines at an effector totarget (E:T) ratio of 8:1 (1×10⁵:1.25×10⁴) for 24 hours and MSDimmunoassay was performed to evaluate the concentration of cytokinessecreted. Cytokine concentrations were determined for IL-2 (FIG. 20A)IFN7 (FIG. 20B) and TNFα (FIG. 20C) following cocultures with OVCAR-3 orOVCAR3-OKT3 cell lines. Non-treated T cells were used as a control. Theresults represent 1-3 biological replicates with 3 technical replicateseach.

Nontransduced (MOCK) and anti-MSNL CoStAR transduced HD T cells werecocultured with engineered K562 target cell lines at an effector totarget (E:T) ratio of 8:1 (1×10⁵:1.25×10⁴) for 24 hours and MSDimmunoassay was performed to evaluate the concentration of cytokinessecreted. Cytokine concentrations were determined for IL-2 (FIG. 21A)IFN7 (FIG. 21B) and TNFα (FIG. 21C) following cocultures with K562-MSNLor K562-MSNL-OKT3 cell lines. Non-treated T cells were used as acontrol. The results represent 1-3 biological replicates with 3technical replicates each.

Anti-CEA CoStAR Expression

Anti-CEA CoStAR expression was evaluated for anti-CEA CoStARs comprisingdiffering signal peptides (Table 11) or scFv antigen binding domains(Table 12).

TABLE 11 Clone CTP194 CTP255 CTP256 CTP257 CTP258 CTP259 SEQ ID NO: 4243 44 45 46 47 Signal peptide OSM1 CD8 CD2 IL2 GMCSF hIgGκ scFv MFE23Linker AAAGSGGSG Spacer CD28 EC TM CD28 Intracellular CD28.CD40

TABLE 12 Clone CTP194 CTP219 CTP220 CTP221 CTP222 CTP223 SEQ ID 42 60 7896 114 132 NO: Signal OSM1 peptide scFv MFE23 MFE23 hMFE23 CEA6 BW431/26hT84.66 (Q > K) Linker AAAGSGGSG Spacer CD28 EC TM CD28 Intra- CD28.CD40cellular

To examine signal peptide variants, T cells from healthy donor PBMCswere lentivirus transduced at a multiplicity of infection (MOI) 5, withthe MFE23 scFV constructs against the carcinoembryonic antigen 5 (CEA)that possessed CD28.CD40 domains. The constructs had variations in thesignal peptide and non-transduced (MOCK) cells were used as controls.Cells were sorted using CD34 microbeads and underwent a rapid expansionprotocol (REP) for 14 days. Following expansion, 1×10⁵ cells wereassessed for transduction efficiency (FIG. 22 ) either via surfacedetection of the marker gene tCD34 or CoStAR molecule using ananti-CD34-APC (black bars) or using a primary rhCEACAM5-Fc antibody witha secondary anti-IgG-Fc-PE (grey bars) antibody, respectively.

T cells from healthy donor PBMCs were also lentivirus transduced, at amultiplicity of infection (MOI) 5, with variable scFV constructs againstthe carcinoembryonic antigen 5 (CEA) that possessed CD28.CD40 domains.As above, non-transduced (MOCK) cells were used as controls. Cells weresorted using CD34 microbeads and underwent a rapid expansion protocol(REP) for 14 days. Following expansion, 1×10⁵ cells were assessed fortransduction efficiency (FIG. 23 ) either via surface detection of themarker gene tCD34 or CoStAR molecule using an anti-CD34-APC (black bars)or using a primary rhCEACAM5-Fc antibody with a secondary anti-IgG-Fc-PE(grey bars) antibody, respectively.

Example 10 Anti-CEA CoStAR Activity

Cytokine production was assessed in CoStAR transduced HD T cellscocultured with Lovo target cell lines. CoStARs comprising differentanti-CEA binding domains (Table 12) were tested. Nontransduced (MOCK)and anti-CEA CoStAR transduced HD T cells were cocultured withengineered target cell lines at an effector to target (E:T) ratio of 8:1(1×10⁵:1.25×10⁴) for 24 hours and MSD immunoassay was performed toevaluate the concentration of cytokines secreted. Cytokineconcentrations were determined for IL-2 (FIG. 24A) IFN7 (FIG. 24B) andTNFα (FIG. 24C) following cocultures with Lovo or Lovo-OKT3 cell lines.Non-treated T cells were used as a control. The results represent 1biological replicate with 3 technical replicates.

Cytokine production was also assessed in CoStAR transduced HD T cellscocultured with K562 target cell lines. As above, cells were coculturedwith engineered target cell lines at an effector to target (E:T) ratioof 8:1 (1×10⁵:1.25×10⁴) for 24 hours and MSD immunoassay was performedto evaluate the concentration of cytokines secreted and cytokineconcentrations were determined for IL-2 (FIG. 25A) IFN7 (FIG. 25B) andTNFα (FIG. 25C) following cocultures with K562.CEACAM5 orK562.CEACAM5.OKT3 cell lines. Non-treated T cells were used as acontrol.

Spacer-transmembrane variants were also examined. In one experiment,anti-CEA CoStARs comprising an hMFE23 CEA-binding domain and differentspacer/transmembrane domains (Table 13) were compared.

TABLE 13 Clone CTP220 CTP232 CTP244 SEQ ID NO: 78 84 162 Signal PeptideOSM1 scFv hMFE23 linker AAAGSGGSG Spacer CD28 EC CD8 EC CD28 (trunc IIH)TM CD28 CD8 CD28 Intracellular CD28.CD40

T cells from healthy donor PBMCs were lentivirus transduced at amultiplicity of infection (MOI) 5, with the hMF23 scFV constructsagainst the carcinoembryonic antigen 5 (CEA) that possessed CD28.CD40domains. Cells were sorted using CD34 microbeads and underwent a rapidexpansion protocol (REP) for 14 days. Following expansion, 1×10⁵ cellswere assessed for transduction efficiency (FIG. 26 ) via surfacedetection of the marker gene tCD34 using an anti-CD34-APC (black bars)or detection of the CoStAR molecule or using a primary rhCEACAM5-Fcantibody with a secondary anti-IgG-Fc-PE (grey bars) antibody. Allvariants were efficiently expressed.

Cytokine production was assessed in CoStAR transduced HD T cellscocultured with Lovo target cell lines. As above, cells were coculturedwith engineered target cell lines at an effector to target (E:T) ratioof 8:1 (1×10⁵:1.25×10⁴) for 24 hours and MSD immunoassay was performedto evaluate the concentration of cytokines secreted and cytokineconcentrations were determined for IL-2 (FIG. 27A) IFN7 (FIG. 27B) andTNFα (FIG. 27C) following cocultures with Lovo or Lovo-OKT3 cell lines.Non-treated T cells were used as a control.

Example 11

CoStARs were constructed to test intracellular signaling domains. FIG.28 and Table 14 depict anti-CEA CoStARs comprising an hMFE23 CEA-bindingdomain with intracellular signaling domains comprising CD40, CD134,CD137, CD2, ICOS, DAP10, and NTRK1 signaling elements.

TABLE 14 Clone CTP313 CTP314 CTP315 CTP316 CTP317 SEQ ID NO: 344 345 346347 348 Signal peptide OSM1 scFv hMFE23 Linker AAAGSGGSG Spacer CD28 ECICOS TM CD28 ICOS Intracellular NTRK1 NTRK1 CD28 CD28 ICOS CD40 NTRK1NTRK1 CD40 CD40 Clone CTP318 CTP319 CTP320 CTP321 CTP322 CTP323 SEQ IDNO: 349 350 351 352 353 354 Signal peptide OSM1 scFv hMFE23 LinkerAAAGSGGSG Spacer CD28 EC CD2 EC CD28 EC TM CD28 CD2 CD28 IntracellularCD28 CD28 CD2 CD28 CD28 CD28 ICOS ICOS CD40 CD2 CD40 CD137 CD40 CD2Clone CTP324 CTP325 CTP326 CTP327 CTP328 SEQ ID NO: 355 356 357 358 359Signal peptide OSM1 scFv hMFE23 Linker AAAGSGGSG Spacer CD28 EC TM CD28Intracellular CD28 CD28 CD28 CD28 CD28 CD40 DAP10 CD40 CD134 CD40 CD137DAP10 CD134

T cells from healthy donor PBMCs were lentivirus transduced at amultiplicity of infection (MOI) 5 with the hMF23 scFV constructs againstthe carcinoembryonic antigen 5 (CEA). Nontransduced (MOCK) cells wereused as controls. Cells were sorted using CD34 microbeads and underwenta rapid expansion protocol (REP) for 14 days. Following expansion, 1×10⁵cells were assessed for transduction efficiency (FIG. 29 ) either viasurface detection of the marker gene tCD34 or CoStAR molecule using ananti-CD34-APC (black) or using a primary rhCEACAM5-Fc antibody with asecondary anti-IgG-Fc-PE (red) antibody, respectively. The resultsrepresent 3 biological replicates.

CoStAR transduced cells were phenotypically characterized. Followingoutgrowth and REP, 1×10⁵ cells were assessed for the differentiationsubtype using flow cytometry.

TABLE 15 T Cell Differentiation Subtype Definition TN CD45RO− CCR7+CD95− Tscm CD45RO− CCR7+ CD95+ Tcm CD45RO+ CCR7+ CD95+ Tem CD45RO+ CCR7−CD95+ Tte CD45RO− CCR7− CD95+ Tcm, central memory T cell; Tem, effectormemory T cell; Tn, naïve T cell; Tscm; stem cell memory T cell; Tte,terminal effector T cell

T cells from HD PBMCs of three donors were lentivirus transduced withthe hMF23 scFV constructs of FIG. 28 . Cells were sorted using CD34microbeads and underwent a rapid expansion protocol (REP) for 14 days.Following outgrowth and REP, 1×10⁵ cells were assessed for thedifferentiation subtype compared to non-transduced cells using flowcytometry. T cell phenotypes are depicted in FIG. 30 as a proportion ofCD3 cells.

Example 12

Cytokine secretion anti-CEA hFME23 CoStAR transduced T cells wasassessed by coculture with K562 target cells. Nontransduced (MOCK) andanti-CEA CoStAR transduced HD T cells were cocultured with engineeredtarget cell lines at an effector to target (E:T) ratio of 8:1 (1×10⁵:1.25×10⁴) for 24 hours and MSD immunoassay was performed to evaluate theconcentration of cytokines secreted. Cytokine concentrations for IL-2(FIG. 31A) IFN7 (FIG. 31B) and TNFα (FIG. 31C), following cocultureswith K562.CEACAM5 (signal 2) or K562.CEACAM5.OKT3 (signal 1+2) celllines are shown. Non-treated T cells were used as a control.

Cytokine expression was also assessed. Nontransduced (MOCK) and anti-CEACoStAR transduced HD T cells were cocultured with engineered target celllines at an effector to target (E:T) ratio of 1:1 (1×10⁵: 1×10⁵) for 16hours in the presence of Brefeldin A and cytokine producing cells weremeasured using intracellular flow cytometry. Frequency of IL-2 (FIG.32A) IFN7 (FIG. 32B) and TNFα (FIG. 32C) expressing cells followingcocultures with K562.CEACAM5 (signal 2) or K562.CEACAM5.OKT3 (signal1+2) cell lines are shown. Non-treated T cells were used as a control.

Example 13 Proliferation of Anti-CEA CoStAR Transduced Cells

HD T cells were transduced with hMFE23 anti-CEA CoStARs and coculturedwith engineered target cells. Nontransduced (MOCK) and anti-CEA CoStARtransduced HD T cells were cocultured with K562.CEACAM5.OKT3 engineeredtarget cell lines at an effector to target (E:T) ratio of 8:1 (1×10⁵:1.25×10⁴) on Day 0. Nontransduced (MOCK) cells were used as controls. OnDay 7, a maximum 50000 cells were re-stimulated with K562.CEACAM5.OKT3engineered target cell lines at an E:T of 8:1. On Day 5, Day 7 and Day 9post stimulation a portion of the cocultures were collected for countingby flow cytometry. For all counts, DRAQ7 was used for live celldiscrimination and CD2 to enumerate the T cells (FIG. 33 ). The figuresrepresent fold expansion of input cells. N=2

HD T cells transduced with hMFE23 anti-CEA CoStARs and cocultured withK562.CEACAM5.OKT3 engineered target cell lines as above were sampled forcounting on Day 6 to evaluate fold expansion (FIG. 34 ). Cells werecounted by flow cytometry using DRAQ7 for live cell discrimination andCD2 to enumerate the T cells.

Example 14

Signal Transduction and Intracellular Domain Binding Sites and Motifs.

The effect of mutations in TRAF2/TRAF3 and TRAF6 binding sites (FIG. 35) of CD40 on cytokine secretion and long term survival and proliferationof CD28.CD40 CoStAR transduced T cells was examined. Cells of threedonors were activated with Dynabeads and transduced with WT CD28.CD40(CTP194), CD28.CD40 containing TRAF2 binding site mutation SVQE>AVQA(CTP195), TRAF2/TRAF3 binding site mutation PVQET>AVAEA (CTP196), TRAF6binding site mutation PQEINF>AQAINF (CTP197), Q263A (CTP199), or mocktransduced. Cells were enriched for CD34 marker expression, expandedfollowing the rapid expansion protocol (REP) and frozen for subsequentexperiments. After thaw, cells were rested for 3-4 days in complete RPMIsupplemented with IL-2 and their transduction rate was determinedlooking at the CD34 marker gene expression. The viability and absolutecount were assessed after overnight IL-2 starvation using DRAQ-7 (1:200)by flow cytometry (Novocyte) and data were analysed using theNovoExpress 1.5.0 software. Transduced T cells were cocultured inabsence of IL-2 with LoVo (CCL-229™ from ATCC) or LoVo.OKT3.GFP tumorcells at 8:1 effector to target ratio. After 24 hours, supernatants werecollected and frozen. LoVo and LoVo.OKT3.GFP naturally express CEA andPD-L1 on their surface, conferring signal 2 through the CoStAR alone(LoVo) or associated with signal 1 (LoVo.OKT3.GFP) to the transduced Tcells. Cocultures were performed in triplicates and correspondingnegative (T cells alone, tumor cells alone) and positive (PMA+ionomycin)controls were included in the experiment. After thaw, secreted IL-2 wasdetected by ELISA and the absorbance was measured using the FLUOstarOmega microplate reader and subsequently analysed with the Omega MARS3.42 R5 software (FIG. 36A). Each dot represents the mean of triplicatesfor one donor. Note that negative controls (T cells alone, tumor cellsalone) were all below the detection range.

After 6-8 days, the viability and absolute count were assessed, and liveT cells were rechallenged for an additional week with freshLoVo.OKT3.GFP tumor cells as described above. At the end of thelong-term coculture, the viability and absolute count were measured, andthe fold expansion was calculated (FIG. 36B). Data shown as mean+/−SEMof n≤3 donors analysed in triplicates.

Mutation of the TRAF2 binding site (SVQE>AVQA; CTP195) resulted littlereduction in IL-2 secretion and moderate reduction of expansion.Mutation of the TRAF2/TRAF3 binding site (PVQET>AVAEA; CTP196) resultedin substantial reduction in IL-2 secretion and expansion. Mutation ofthe TRAF6 binding site (PQEINF>AQAINF; CTP197) resulted in moderatereduction in IL-2 secretion and moderate reduction of expansion.

Example 15

Non-transduced (Non-Td) and anti-FOLR1 CoStAR transduced (Td) TILs weregenerated using a 24-day protocol. Briefly, aliquots of OC digest werethawed and transduced with anti-FOLR1 CoStAR lentivirus at amultiplicity of infection (MOI) of 5 at 48 h and 72 h. Cells were thenexpanded for 8 days (outgrowth), and then subjected to a rapid expansionprotocol (REP) with allogeneic irradiated peripheral blood mononuclearcells (PBMCs) for 12 days.

After production, TIL CD4/CD8 ratio and anti-FOLR1 CoStAR transductionefficiency was measured. TILs were phenotypically characterized fortheir differentiation status, expression of co-inhibitory andco-stimulatory markers, T cell subsets and cytokine producing potentialusing flow cytometric panels.

Complete TIL T cell media (TCM) for outgrowth consists of 450 mL ofGIBCO custom P158718 media with 50 mL of heat inactivated Fetal BovineSerum, gentamycin (10 μg/mL)/amphotericin (0.25 μg/mL) and vancomycin(50 μg/mL). Complete rapid expansion protocol (REP) media consists of460 mL of GIBCO custom P158718 with 40 mL human AB serum, gentamycin (10μg/mL)/amphotericin (0.25 μg/mL) and vancomycin (50 μg/mL).

Generation of Anti-FOLR1 CoStAR OC TILs

Five OC samples were used to generate anti-FOLR1 CoStAR modified TILs.The outgrowth period of TILs was 12 days. On day 1 (D1), samples fromeach donor were thawed in complete TIL TCM, the cells were washed onceby centrifuging at 400×g for 5 minutes, resuspended in fresh TIL TCM andcounted. All cell counts were performed using a DRAQ7 dye and anti-CD2antibody stains using a Novocyte 3005 Flow Cytometer System. TILs werethen centrifuged at 400×g for 5 minutes, resuspended at a concentrationof 1×10⁶ cells/mL, placed into an appropriate vessel with 3000 IU/mLIL-2 and rested for two days in a 5% CO2 incubator set to 37° C.

Following the rest period on day 3, cells were collected, washed,centrifuged at 400×g for 5 minutes, and resuspended in fresh completeTCM. The number of viable cells in each sample was determined using aNovocyte 3005 as described above, cells were centrifuged at 400×g for 5minutes and resuspended at a concentration of 1×10⁶ cells/mL. Eachsample was split into two equal parts, one for production of Non-Td andother for transduced (Td) TIL, modified to express anti-FOLR1 CoStAR.Transduction with anti-FOLR1 CoStAR lentivirus was performed at an MOIof 5 based on the total number of live cells. IL-2 was added at aconcentration of 3000 IU/mL and the cells were placed in a 5% CO2incubator set to 37° C.

On day 4, the cells were collected, washed once with complete TIL TCM,and resuspended in the same volume of fresh complete TIL TCM as on day 3for the second day of transduction. Transduction was performed using theanti-FOLR1 CoStAR lentivirus at an MOI of 5 and IL-2 at 3000 IU/mL wasadded to the cells prior to placing them in a 5% CO2 incubator set to37° C. for 8 days. IL-2 (3000 IU/mL) was added to the cells every 2-3days until D13.

On day 13 cells were collected, washed, resuspended in complete TIL TCMand counted using a Novocyte 3005. After determining the TIL numbers onday 13, the cells were seeded in appropriate scale G-REX plates for REPusing 10 healthy donors worth of irradiated allogeneic PBMCs as feedersat a 200:1 ratio of feeders:TIL. The media used for the REP was thecomplete REP TIL TCM with anti-CD3 (OKT3) antibody was added at aconcentration of 30 ng/mL for activation. IL-2 was added at aconcentration of 3000 IU/mL and the cells were placed in a 5% CO2incubator set to 37° C. The REP period was 12 days during which IL-2(3000 IU/mL) was supplemented every 2-3 days.

On day 19 (ie, day 6 of REP), 5 mL of medium from the 24 well G-REXplates or 25 mL of medium from the 6 well G-REX plates was removedwithout disturbing the cells and replaced with fresh complete REP TILTCM and IL-2 (3000 IU/mL). On D25, at the end of the REP, TILs wereharvested by centrifugation at 400×g for 5 minutes and resuspended infresh media for counting using a Novocyte 3005. TILs were resuspended ata concentration of 1×10⁶ cells/mL. TILs were then assessed fortransduction efficiency by staining 1×10⁵ cells of each sample withantibodies against CD3, CD4, CD8, CoStAR and a viability stain. DNA wasextracted from 1×10⁶ cells from each sample using the DNeasy Blood &Tissue Kit following the manufacturer's instructions. Isolated DNA wasused to analyze the vector copy number (VCN) using Droplet Digital PCR(ddPCR) and primers specific to the anti-FOLR1 CoStAR and the referencegene Poly(rC) binding protein 2 (PCBP2). For subsequent experiments2-5×10⁷ cells were rested in fresh complete REP TIL TCM for 3 days withIL-2 (3000 IU/mL). Remaining TIL were resuspended in cryoprotectant andaliquoted to cryovials, cooled to −80° C. overnight, and thentransferred to −150° C. for short term storage. Cryopreserved TIL werethawed in a 37° C. water bath, washed once with PBS by centrifugation at400×g for 5 minutes, then underwent an identical rest period asdescribed above prior to experimentation.

Phenotypic Characterization of Anti-FOLR1 CoStAR OC TILs from FourDonors.

Non-Td and Td cells from four donors were rested for 3 days in REP TCMmedia with IL-2 (3000 IU/mL). Subsequently, the cells were harvested,washed once with media by centrifugation at 400×g for 5 minutes,resuspended in fresh complete REP TIL TCM, counted using Novocyte 3005,and resuspended at a concentration of 1×10⁶ cells/mL. Cytometricevaluation of TILs was performed on 1×10⁵ cells per well, intriplicates, using four flow cytometry panels. Assessment ofdifferentiation status was performed using a panel with antibodiesagainst CD3, CD4, CD8, CD27, CD95, CCR7, CD45RA, CD45RO, CoStAR, and aviability stain. Coinhibitory and costimulatory marker expression wasassessed using antibodies against CD4, CD8, CD137, programmed cell deathprotein 1 (PD-1), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4),lymphocyte-activation gene 3 (LAG-3), T cell immunoglobulin and mucindomain protein 3 (TIM-3), signaling lymphocyte activation molecule(SLAM), CoStAR, and a viability stain. Cell subpopulations, includingTregs, were assessed using antibodies against CD3, CD4, CD25, forkheadbox Protein 3 (FOXP3), T cell receptor alpha beta (TCRαβ), T cellreceptor gamma delta (TCRγδ), CD56, CD127, CoStAR, and a viabilitystain. Cytokine production upon mitogenic stimulation was assessed usingantibodies against CD3, CD4, CD8, IL-22, TNFα, IL-17A, IFNγ, CoStAR, anda viability stain. For this panel, TILs were activated by addition ofPMA (50 ng/mL)/ionomycin (1 μg/mL), and Brefeldin A (1000×) and placedin a 5% CO2 incubator set to 37° C. for four hours. Followingactivation, TILs were collected, centrifuged at 400×g for 5 minutes,counted, and 1×10⁵ cells were seeded in triplicate for cytometricanalysis. For all flow cytometry panels, fixation and permeabilizationwas performed using BD Cytofix/Cytoperm per manufacturer's instructions.Following staining, cells were washed and resuspended in PEF (500 mLDPBS, 2 mL EDTA and 2.5 mL of heat inactivated FBS) for analysis using aNovocyte 3005 Flow Cytometer System.

Analysis of the Non-Td and Td Cells

For the four panels of phenotypic characterization, recombinant humanFOLR1 with Fc tag (rhFOLR1-FC) was used for the detection of anti-FOLR1CoStAR cells, and the populations of interest were reported from CoStAR−subset for the Non-Td TILs, and both CoStAR− and CoStAR+ fractions fromthe Td TILs (anti-FOLR1 CoStAR− Td TILs and anti-FOLR1 CoStAR+Td TILs).Further subset characterization was performed on these populations.

Differentiation Status

The gating strategy employed for characterizing T cell differentiationstatus was as follows: a lymphocyte gate followed by doublet and deadcell exclusion, CD3+, CD4+ and CD8+ gates. From all three populations(CD3+, CD4+, and CD8+), further analysis was performed on the T cellfractions of interest. To characterize the different T cell memorysubsets, CD45RA+CD45RO− and CD45RA-CD45RO+ cells were gated from CD3+cells. CD45RO+CCR7+ and CD45RO+CCR7− populations were then gated fromCD45RA-CD45RO+ cells. Using these gates, the central memory T cells(Tcm; CD45RO+CCR7+CD95+CD27+) cells and effector memory T cells (Tem;CD45RO+CCR7−CD95+CD27+/−) cells were further gated. Additionally,CD45RA+CCR7+ and CD45RA+CCR7− populations were gated from CD45RA+CD45RO−cells. Using these gates, stem cell memory T cells (Tscm;CD45RA+CCR7+CD95+CD27+) and naïve T cells (Tn; CD45RA+CCR7+CD95−CD27+)were gated from CD45RA+CCR7+ cells, whilst terminal effector T cells(Tte; (CD45RA+CCR7−CD27−CD95+) cells were gated from CD45RA+CCR7− cells.

Coinhibitory and Costimulatory Markers

The gating strategy employed for characterizing coinhibitory andcostimulatory molecules included doublet and dead cell exclusion. Gatingof CD4+ and CD8+ and CoStAR+/− cells was performed and populations werefurther analyzed for CD137, PD-1, CTLA-4, LAG-3, TIM-3 and SLAMexpression.

T Cell Subtype

The gating strategy employed for characterizing T cell subtypes includeddoublet and dead cell exclusion and a CD3+ gate. Using thesepopulations, the expression of TCRαβ, TCRγδ, and CD56 was assessed.Subsequently, the CD3+CD4+ cells were gated for expression of TCRαβ andCD56, and further analysis of the CD3+CD4+TCRαβ+ population for CD25 andCD127 expression was performed. Using the CD25+CD127− gate the FOXP3+cells were gated to determine the population of Tregs. Therefore, Tregsare defined as CD3+TCRab+CD4+ CD25+CD127-FOXP3+ and CoStAR+/− dependingon the population assessed.

Cytokine Production Upon Mitogenic Stimulation

The gating strategy employed for characterizing intracellular cytokineproduction included doublet and dead cell exclusion, CD3+, CD4+, andCD8+ gates. CoStAR expression analysis on the different populations wasperformed followed by further analysis of the frequency of cellsexpressing IL-22, IL-17A, TNFα, and IFN7.

Clinical Characteristics

Clinical characteristics of TILs from five ovarian cancer patients areshown in Table 16. Patients were all treatment naïve, and the specifichistology revealed three serous cystadenocarcinomas, one endometrioid,and one clear cell adenocarcinoma. Sample weights ranged between0.55-2.62 grams with a mean weight of 2.39±1.31 grams. After processing,samples were cryopreserved at 6×10⁶-2.5×10⁷ cells in 1 mL per vial. Onday 1, samples were thawed and counted using flow cytometry where theaverage percentage of CD2+ TILs was 19.5±9.43. The total TILs harvestedin the thawed samples ranged between 4.4×10⁵-3.3×10⁶ cells.

TABLE 16 Ovarian tumor sample information Tumor type/ Tumor ClinicalTreatment ID Histology weight Markers stage TNM Grade status 1 OvarianCancer/ 2.62 CA125- IB T1b- G2-Moderately Treatment- Serous 937 N0-M0differentiated naïve cystadenocarcinoma- M-84413 2 Ovarian Cancer/ 0.55CA125- IV T3-Nx- G3-Poorly Treatment- Serous 478,83 M1 differentiatednaïve cystadenocarcinoma- M-84413 3 Ovarian 4.2 CA125- IIB T2b-G3-Poorly Treatment- Cancer/Serous 50,53 Nx-M0 differentiated naïvecystadenocarcinoma- M-84413 4 Ovarian Cancer/ 2.1 CA125- IC T1c-G2-Moderately Treatment- Endometrioid 1096 Nx-M0 differentiated naïvecarcinoma-M- HE4- 83803 1487 5 Ovarian Cancer/ 2.5 CA125- IIIC T3c-G3-Poorly Treatment- Clear cell 187 Nx-M0 differentiated naïveadenocarcinoma-M- 83103 Abbreviations: ID, identification; CA125, cancerantigen 125; TN M, tumor, nodes, metastases.

After TIL production, cell numbers of Non-Td and Td TILs were comparedto assess any effect of anti-FOLR1 CoStAR modification on TIL growth.Results showed no significant impact on the cell numbers of TILs (FIG.38 ). Proportions of transduced CD3+, CD4+ and CD8+ cells were assessedafter TIL production (FIG. 39A-C). CD4 cells showed higher transductionpercentage (50.8±15.2%) than the CD8 cells (32.7±20.6%), and this wasstatistically significant (p=0.384). Additionally, VCN was assessedusing ddPCR to detect the CoStAR transgene relative to the PCBP2reference gene and <4 integrations per cell were measured (FIG. 39 ).Comparison of the CD4 and CD8 population composition in the productindicated most cells were CD4+ with levels ranging between 53.0-58.7%and fewer CD8+ cells (30.8-38.2%). CD4+CD8+ and CD4−CD8− cells were alsodetected at low levels ranging between 3.47-4.43% and 3.19-6.97%,respectively. There were no significant differences between the Non-Td,anti-FOLR1 CoStAR-Td, and anti-FOLR1 CoStAR+ Td TILs with regards to theCD4/CD8 cell composition (FIG. 40 ).

The TILs were further characterized to determine whether anti-FOLR1CoStAR modification impacted TIL function. The composition of Tn, Tscm,Tcm, Tem and Tte were assessed from CD3+, CD4+ and CD8+ T cellcompartments of Non-Td, anti-FOLR1 CoStAR− Td and anti-FOLR1 CoStAR+ TdTILs (FIG. 41A-C). Results show that majority of TILs were Tem acrossthe CD3+(63.2-78.6%), CD4+(76.9-88.8%) and CD8+(48.3-66.9%) populations.Tcm cell frequencies were between 6.28-15.7%, 6.72-17.5% and 5.18-11.7%for CD3+, CD4+ and CD8+ TILs, respectively. Tte cells made up 14.8-15.9%of bulk CD3+ TILs. These were mainly in the CD8+ subpopulation withfrequencies of 22.1-25.1% versus 2.68-3.79% in the CD4+ subpopulation.Few Tscm cells were detected for CD3+, CD4+ and CD8+ TILs withfrequencies of 0.15-1.38%, 0.05-0.50% and 0.53-3.24%, respectively. Thelowest frequency memory subtype were the Tn cells, and these rangedbetween 0.002-0.019% across all three populations. There were nosignificant differences between the Non-Td, anti-FOLR1 CoStAR− Td andanti-FOLR1 CoStAR+ Td TILs with regards to Tn, Tem, Tcm or Ttefrequencies. Despite the low Tscm TILs, significantly lower Tscm TILswere detected in the anti-FOLR1+Td TILs in CD3+(0.15±0.05% vs1.38±0.69%) and CD4+(0.05±0.02% vs 0.51±0.27%) populations compared tothe anti-FOLR1− Td TILs, but not the Non-Td TILs. Overall, the datashows that anti-FOLR1 CoStAR modification did not significantly affectdifferentiation status of TILs.

CD8+ and CD4+ TILs were assessed for the expression of CD137, PD-1,CTLA-4, LAG-3, TIM-3 and SLAM (FIG. 42 ). In CD4+ TILs, expression forall three populations of interest were in the range of 39.8-47.7%,61.0-64.4%, 52.7-57.6, and 65.6-72.1% for LAG-3, PD-1, SLAM, and TIM-3,respectively. In CD8+ TILs, expression ranged between 86.7-88.2%,38.2-46.5%, 61.5-67.2%, and 90.0-94.3% for LAG-3, PD-1, SLAM, and TIM-3,respectively. CTLA-4 ranged between 8.92-15.2% and 4.10-7.29%, and CD137was 2.80-8.30% and 7.70-17.1%, for CD4+ and CD8+ cells, respectively.

Comparison of the three TIL populations, Non-Td, anti-FOLR1 CoStAR− Tdand anti-FOLR1 CoStAR+ Td TILs, indicated that there was no effect ofthe anti-FOLR1 CoStAR on coinhibitory and costimulatory markerexpression in CD4+ TILs. In CD8+ TILs, CD137+(17.1±8.79% vs 7.70±3.92%vs 7.77±4.01%) and CTLA-4+(7.29±2.41% vs 4.66±1.51% vs 4.10±1.07%) cellswere of higher frequency in anti-FOLR1+Td when compared to both thenon-Td and anti-FOLR1 CoStAR− Td TILs. PD-1+ TIL frequency was onlyhigher in comparison to the anti-FOLR1− Td TILs but not the Non-Td TILs(46.5±21.1% vs 38.2±19.6% vs 40.6±26.8%, respectively). Overall, therewas little observed effect of CoStAR modification of TILs forcoinhibitory or costimulatory marker expression except for the slightbut significant increase in the frequency of CD8+CD137+, CTLA4+, andPD-1+ TILs.

T cell subset frequency was assessed in TIL samples, using markers forTreg detection in addition to the expression of TCRαβ and TCRγδ. Themajority of the CD3+ cells expressed TCRαβ (90.0-93.5%) with relativelyfew TCRγδ cells (1.35-3.08%) detected (FIG. 43A) and no differences wereobserved between the three populations. The percentage of Tregs(CD3+TCRαβ+CD4+CD25+CD127-FOXP3+) either in the CoStAR- or CoStAR+ TILswas very low. Specifically, the detection frequencies were 0.63±0.48%,0.66±0.36% and 0.93±0.63% for non-Td, anti-FOLR1 CoStAR− and anti-FOLR1CoStAR+ Td TILs, respectively (FIG. 43B). Overall, there was no effectof anti-FOLR1 CoStAR on TCRαβ, TCRγδ, and Treg frequencies whencomparing the Non-Td, anti-FOLR1 CoStAR− Td and anti-FOLR1 CoStAR+ TdTILs.

TILs were assessed the ability of the cells to produce cytokines uponmitogenic activation using PMA/ionomycin. The Non-Td and Td TILs wereactivated for 4 hours using PMA/ionomycin and then stained for IFNγ,IL-22, IL-17A, and TNFα from CD3+, CD4+, and CD8+ cells. Uponstimulation, high frequencies of CD3+ TILs expressing TNFα (61.8-73-6%)and IFN7 (32.5-42.0%), and lower frequencies of IL-22 (4.74-8.07%) andIL-17A (5.74-11.0%) expressing cells were detected (FIG. 44A). Thefrequency of TNFα+ cells was higher in anti-FOLR1 CoStAR+ Td compared toanti-FOLR1 CoStAR− Td TILs (73.6±14% vs 61.8±18.3%), but not Non-Td TILs(72.1±9.20%).

In CD4+ TILs, high frequencies of cells expressing TNFα (54.3-67.2%),and lower frequencies of IFN7 (19.3-26.2%), IL-22 (7.63-10.5%) andIL-17A (12.0-20.2%) expressing cells were detected (FIG. 44B). Thefrequencies of TNFα+(67.2±15.5% vs 54.3±17.2%) and IL-17A+ cells(20.2±11.3 vs 12.0±7.06) were higher in anti-FOLR1 CoStAR+ Td relativeto anti-FOLR1 CoStAR− Td TILs. No significant differences were observedin the frequency of CD4+ expressing TNFα or IL-17A when comparing eitheranti-FOLR1 CoStAR+/−Td TILs to Non-Td TILs.

Similar observations were made for the CD8+ TIL population, where highTNFα (62.7-86.7%) and IFN7 (45.3-63.0%), and lower IL-22 (6.80-17.0%)and IL-17A (6.93-15.3%) frequencies of positive cells were detected(FIG. 44C). In this population, higher frequencies of cytokinesexpressing cells were identified in the anti-FOLR1 CoStAR+ Td TILscompared to anti-FOLR1 CoStAR− TILs with regards to TNFα (86.7±8.37% vs62.7±18.2%), IL-17A (15.3±7.24% vs 6.93±2.77%), and IL-22 (17.0±6.90% vs6.80±2.48%). Again, no significant differences were observed betweeneither anti-FOLR1 CoStAR+/−Td TILs compared to Non-Td TILS.

The significant differences observed both in the CD4+ and CD8+subpopulations between the IL-17A+ anti-FOLR1 CoStAR+ Td TILs andanti-FOLR1 CoStAR-Td TILs were not statistically significant in the CD3+bulk population (p=0.0527). Collectively, some statistically significantdifferences were observed within CD3+, CD4+, or CD8+ TIL populations inthe proportion of IL-17A, IL-22, and TNFα expressing cells betweenanti-FOLR1 CoStAR+/− populations of Td cells. However, no significantdifferences were observed in the frequency of IFNγ, IL-17A, IL-22, andTNFα producing cells when comparing either Td population (ie, anti-FOLR1CostAR −/+ fractions) to Non-Td TILs.

Example 16 Antitumor Reactivity of CoStAR Positive TILs

Complete rapid expansion protocol (REP) medium consisted of 460 mL GIBCOcustom P158718 supplemented with 40 mL human AB serum, gentamycin (10μg/mL)/amphotericin (0.25 μg/mL) and vancomycin (50 μg/mL). Complete Tcell medium (TCM) consisted of 450 mL RPMI 1640 GlutaMAX™ SupplementHEPES medium supplemented with 5 mL Penicillin-Streptomycin, 500 μL2-Mercaptoethanol (50 mM), and 50 mL Fetal Bovine Serum (FBS).

Preparation of CoStAR Modified OC TILs for Functional Characterization

Non-Td and Td TILs from five OC samples were produced as described inITIL-306-NC-010 and the transduction percentages are shown in Table 17.

TABLE 17 Non-Td and Td TIL donors CoStAR CoStAR % of % of CD3 in CD3Donor Non-Td in Td Symbol 1 0.98 34.00 ▪ 2 0.11 24.33 ● 3 0.20 47.11 ▾ 40.09 45.65 ▴ 5 0.15 64.60 ♦

TILs were either used directly after the REP or upon thaw from long-termcryopreservation (−150° C.; stored in cryoprotectant consisting of FBSwith 10% DMSO). ovarian cancer TILs were thawed using a 37° C. waterbath, transferred to a 50 mL Falcon tube with 10 mL of complete REP TCM,centrifuged at 400×g for 5 minutes and resuspended in complete REP TCM.Both post-REP and post-thaw TILs, were counted using the NovoCyte 3005Flow Cytometer System following DRAQ7 dead cell exclusion and a CD2+count and placed in T75 flasks at a density of 1×10⁶ cells/mL incomplete REP TCM supplemented with 3000 IU/ml of interleukin 2 (IL-2).The cells were then placed in a 5% CO2 incubator set to 37° C. for2-3-days. Before the assay, TILs were resuspended in complete REP TCM ata density of 1×10⁶ cells/mL without IL-2 and placed in a 5% CO2incubator set to 37° C. overnight.

Assessment of FOLR1 Expression by Autologous Tumor Digest.

All autologous tumor digests were thawed from long-term cryopreservation(−150° C.; stored in cryoprotectant consisting of FBS with 10% DMSO)using a 37° C. water bath, transferred to a 15 mL Falcon tube with 9 mLof complete TCM, centrifuged at 400×g for 5 minutes and resuspended in 5mL of complete TCM. The number of viable cells in each sample wasdetermined using the NovoCyte 3005 Flow Cytometer System following DRAQ7dead cell exclusion, and the cells were resuspended at a concentrationof 1×10⁶ cells/mL in completed TCM. 1×10⁵ autologous tumor digest cellswere then analyzed according to the flow cytometry staining protocol andacquired using the Novocyte 3005 Flow Cytometer System.

The quantification of FOLR1 expression was conducted according to thefollowing gating strategy: a cell gate (forward scatter-height [FSC]-Hvs side scatter-height [SSC]-H), then a doublet exclusion gate (SSC-H vsside scatter-area [SSC-A]) followed by a dead cell exclusion gate (SSC-Avs Fixable Viability dye eFluor 450). The CD2-cells were then gated fromwhich the frequency of the FOLR1+ cells were quantified (SSC-A vsFOLR1-PE).

Cytokine Production Assessed by Intracellular Flow Cytometry

To measure the frequencies of cytokine producing T cells, Non-Td andanti-FOLR1 CoStAR Td TILs were cocultured at a 1:1 effector to targetratio (E:T, 1×10⁵ TILs:1×10⁵ target cells) with either autologous tumordigests, K-562, or OVCAR-3 derived engineered cell lines as targets.Cocultures took place in 96 well round bottom plates with 200 μLcomplete TCM supplemented with 1× Brefeldin A and were incubated in a 5%CO2 incubator set to 37° C. for 16 hours. Unstimulated TILs or targetcells alone were used as negative controls, and positive control TILswere activated with 50 ng/mL phorbol-myristate-acetate (PMA) and 1 μg/mLionomycin. All conditions were performed in triplicates.

Measurement of cytokine production was performed using a panel withantibodies against CD3, CD4, CD8, CoStAR (anti-idiotype 19.1 primary andanti-mouse IgG1 secondary antibodies), tumor necrosis factor alpha(TNFα), IL-2, and a viability stain. Following the 16-hour incubation,cocultures were harvested by centrifugation at 500×g for 4 minutes. Thesupernatant was discarded, and samples were analyzed by flow cytometry.Briefly, cells were labelled using 100 l of fixable viability dye(1:1000 diluted in PBS) and incubated for 10 minutes at roomtemperature. Subsequently, cells were washed using BD stain buffer,centrifuged at 500×g for 4 minutes, and cell pellets were resuspended in100 μl of BD stain buffer with FcR blocking reagent for 10 minutes atroom temperature. Following the incubation step, cells were washed oncewith BD stain buffer and fixed using 4% paraformaldehyde (PFA) for 15minutes at room temperature. A wash with BD perm/wash buffer, stainingusing the 19.1 anti-idiotype for 25 minutes at 4° C. and two more BDperm/wash buffer washes followed. Cells were then resuspended in 100 μlof BD stain buffer with CD3, CD4, CD8, anti-mouse IgG1, TNFα, and IL-2and incubated at 4° C. for 25 minutes. Following two more wash stepsusing BD perm/wash buffer, cell pellets were resuspended in 100 μl of BDstain buffer for acquisition on NovoCyte 3005 Flow Cytometer System.

The quantification of TNFα and IL-2 producing cell frequencies in theCD4 and CD8 subpopulations was conducted according to a gating strategythat included dead cell exclusion. CD3+ cells were gated from the livegate (SSC-A vs CD3-A) and then CD4+ and CD8+ cells were gated from theCD3+ gate (CD4-A vs CD8-A). TNFα and IL-2 producing cells were reportedfrom the CoStAR negative (−) subset for the Non-Td TILs, and bothCoStAR− and CoStAR positive (+) fractions from the Td TILs (anti-FOLR1CoStAR− Td TILs and anti-FOLR1 CoStAR+ Td TILs).

Cytokine Production Assessed by MSD Immunoassay

To measure cytokine secretion, non-Td and anti-FOLR1 CoStAR Td TILs werecocultured at a 1:1 ratio (E:T, 1×105 TILs:1×10⁵ target cells) withautologous tumor digests and at a 8:1 ratio (E:T, 1×10⁵ TILs:1.25×10⁴target cells) with K-562 or BA/F3 derived engineered cell lines.Cocultures were performed in 200 μL complete TCM in triplicate. Inexperiments where MHC blocking was conducted, autologous tumor digestswere pre-incubated (4° C.) with antibodies directed against MHC Class I(HLA-ABC; 40 μg/mL), MHC Class II (HLA-DRDPDQ; 40 g/mL), MHC Class I+II(both 40 μg/mL), or an isotype control (Mouse IgG2a; 80 μg/mL) for 45minutes in 100 μL complete TCM. Following the incubation, TILs wereadded to the appropriate wells. For all experiments, unstimulated TILsor target cells alone were used as negative controls, and TILs activatedwith 50 ng/mL phorbol-myristate-acetate (PMA) and 1 g/mL ionomycin wereused as a positive control. The cells were then placed in a 5% CO2incubator set to 37° C. for 24 hours.

Following the 24-hour incubation, samples were centrifuged at 400×g for5 minutes, and the supernatant was harvested before storage at −80° C.Upon thaw, samples were appropriately diluted in complete TCM andDiluent 2 from the V-Plex Plus Proinflammatory Panel 1 kit from Mesoscale discovery (MSD). The assay was performed per manufacturer'sinstructions.

Target Cell Cytotoxicity Assessed by Flow Cytometry

To measure the cytotoxic activity of TILs, Non-Td and anti-FOLR1 CoStARTd TILs were cocultured at a 1:1 ratio (E:T, 1×10⁵ TILs:1×10⁵ targetcells) with BA/F3 derived engineered cell lines. Cocultures wereperformed in 96 well round bottom plates with 200 μL complete TCM intriplicate, and incubated for 20 hours in a 5% CO2 incubator set to 37°C. TILs and target cells were cultured alone as negative controls.

Following the 20-hour coculture, samples were centrifuged at 400×g for 5minutes, the supernatant was discarded, and samples were analyzed byflow cytometry. Briefly, cells were labelled using 100 μl of fixableviability dye (1:1000 diluted in PBS) and incubated for 10 minutes atroom temperature. All washes were performed using BD stain buffer. Afterthe incubation cells were washed, centrifuged at 500×g for 4 minutes,and cell pellets were resuspended in 100 μl of BD stain buffer with FcRBlocking reagent for 10 minutes at room temperature. Following theincubation step, cells were washed once resuspended in 100 μl of BDstain buffer with CD2 and incubated at 4° C. for 25 minutes. Cells werewashed twice, and cell pellets were resuspended in 100 μl of BD stainbuffer for acquisition on NovoCyte 3005 Flow Cytometer System.

Target cell counts were enumerated by flow cytometry after doublet anddead cell exclusion. CD2-cells were gated from the live gate (SSC-A vsCD2-A) and quantified by the absolute count function of the NovoCyte3005 Flow Cytometer System.

Target Cell Cytotoxicity Assessed by xCELLigence

Assessment of target cell cytotoxicity by xCELLigence was performedaccording to manufacturer's instructions. Briefly, E-plates wereequilibrated by adding 50 μL TCM per well, incubated at room temperaturefor 30 minutes, and background electrical impedance was acquired on theRTCA Analyzer (37° C., 5% CO₂). Following this, 3×10⁴ OVCAR-3 derivedengineered cell lines in 50 μL TCM were added per well of each E-plateand incubated at room temperature for 30 minutes before cell growth wasassessed by electrical impedance (cell index) upon the RTCA Analyzer(37° C., 5% CO₂). The cell index was measured every 15 minutesthroughout the duration of the assay. Upon reaching confluency (between24-31 hours), 6×10³ or 1×10³ Non-Td or Td TILs in 100 μL TCM were addedto OVCAR-3 derived engineered cell lines (E:T ratios of 1:5 and 1:30,respectively). These were incubated at room temperature for 30 minutesprior to 169 hours of further cell index readings upon the RTCA Analyzer(37° C., 5% CO₂). Control conditions included wells containing targetcell lines alone, target cell lines with 0.5% Triton X-100 added at OCTIL loading time-point (full lysis control), and TILs alone. Thenormalized cell index (NCI) was determined according to manufacturer'sinstructions using RTCA software pro. The area under the NCI curve asextracted from RTCA software pro for the 169-hour period of OC TILcoculture with OVCAR-3 derived engineered cell lines is reported as aquantitative readout. Both the RTCA SP and DP were used. For the 1:30ratio of Non-Td and Td TILs from 9831 and 9260 tumor digests, cocultureswere run in duplicate.

Analysis

Following the production and characterization of TIL from cryopreservedtumor samples, retained input tumor digest samples were thawed andassessed for their expression of FOLR1; the ligand of anti-FOLR1 CoStAR.FOLR1 could be detected on the surface of CD2− cells in all 5 tumorswith the proportion of FOLR1 positive cells ranging from 5.44-22.1%(FIG. 45 ).

Intracellular flow cytometry was used to assess the frequency of T cellsthat recognize and respond to autologous tumor. PMA/I stimulation wasused as a positive control for frequencies of TIL capable of readilyproducing IL-2 and TNFα. No IL-2+ or TNFα+ TILs were detected in tumordigests cultured overnight (16 hours) alone with brefeldin A. Incontrast, IL-2+ and TNFα+ TILs were detected upon 16-hour coculture ofexpanded TIL with autologous tumor digest (FIG. 46 ). CD4+ anti-FOLR1CoStAR+ Td TILs were characterized by significantly higher frequency ofIL-2 producing cells compared to Non-Td TILs with a marked 6.56-foldincrease (12.0±12.9% vs 1.83±0.55%) (FIG. 46A). Although notsignificant, CD4+ anti-FOLR1 CoStAR+ Td TILs also had a 3.33-foldincrease in IL-2 producing cell frequencies compared to anti-FOLR1CoStAR− Td TILs (12.0±12.9% vs 3.61±3.10%). A slight but significantincrease in the number of anti-FOLR1 CoStAR+CD4 T cells producing TNFα(6.96±3.56%) when cultured alone relative to anti-FOLR1 CoStAR− Td TILs(2.58±1.60%), but not Non-Td TILs (3.31±3.62%) was also detected (FIG.46C). Upon coculture with the autologous digest, TNFα+ cells seemed toincrease in frequency in the anti-FOLR1 CoStAR+ Td TIL population,although a statistically significant difference compared to anti-FOLR1CoStAR− Td and Non-Td TILs was not observed (32.3% vs 17.8% vs 11.6%).

Similar observations were made for the CD8+ population upon coculturewith autologous digest (FIG. 46A-D). A significantly higher frequency ofCD8+IL-2+ cells was detected by anti-FOLR1 CoStAR+ Td TILs (4.13±2.26%)relative to anti-FOLR1 CoStAR− Td TILs (1.42±1.00%; 2.9-fold increase)but not the Non-Td TILs (1.11±0.55%; 4.13-fold increase, p=0.1097) (FIG.46B). CD8+ TNFα producing cell frequencies were increased in theanti-FOLR1 CoStAR+ Td TILs relative to anti-FOLR1 CoStAR-Td and Non-TdTILs, however, these differences did not reach statistical significance(22.6% vs 12.5% vs 10.5%) (FIG. 46D).

Overall, CoStAR modification significantly increased the frequencies ofCD4+ and CD8+ cells producing IL-2 upon stimulation with the autologousdigest. In the same setting, CD4+ and CD8+ cells frequencies producingTNFα+ trend similarly, although statistical significance was notreached.

An MSD immunoassay was conducted to assess the quantity of cytokinesreleased upon TIL coculture with autologous digest (FIG. 47A-D). PMA/Istimulation was used as a positive control for cytokine production,which was similar between CoStAR modified and Non-Td TIL. CoStARmodification of TIL led to a statistically significant increase in thesecretion of IL-2, TNFα, IL-13 and IFNγ secretion in response toautologous tumor in comparison to Non-Td TILs (FIG. 47A-D). A trend ofincreased background cytokines levels from Td vs Non-Td TILs whencultured alone (FIG. 47A-D) was observed and was only significant forIL-2 (70.6 μg/mL vs 31.8 μg/mL) (FIG. 47A). Upon coculture with theautologous digest, although IL-2 secretion was significantly higher inTd TILs compared to Non-Td TILs (74.1 μg/mL vs 32.1 μg/mL), this was notdifferent from background levels of TILs cultured alone. Td TILssecreted higher levels of IFN7 in response to autologous digest comparedto Non-Td TIL with production levels of 8287 pg/mL vs 1970 μg/mL (FIG.11D). Similarly, higher levels of TNFα (55.0 μg/mL vs 18.2 μg/mL, FIG.47B) and IL-13 (152 μg/mL vs 48.4 μg/mL, FIG. 47C) were detected when TdTILs were cocultured with the autologous digest relative to Non-Td TILs.These indicate a 3-, 4.2- and 3.1-fold increase in TNFα, IFNγ and IL-13production by CoStAR modified Td TILs, respectively.

A statistically significant positive correlation (r²=0.9191) wasobserved between IFN7 released by CoStAR modified TIL upon coculturewith autologous tumor and the proportion of tumor digest expressingFOLR1 (FIG. 47E). This correlation was not observed for Non-Td TILs,confirming CoStAR-FOLR1 interactions are key for TIL enhancement.Moreover, CoStAR modified TIL IFN7 release is enhanced even againstovarian tumors harboring frequencies of FOLR1 positive cells as low as5.44% (FIG. 11E).

MHC blocking antibodies prevent TCRs from engaging their target andmediating TCR signaling. To assess the MHC-restricted antigenrecognition of CoStAR modified TIL, Non-Td and Td TILs were coculturedwith autologous tumor digest in the presence of MHC blocking, orirrelevant isotype control antibodies, and TIL anti-tumor activity wasmeasured by IFN7 release (FIG. 48 ). As CoStAR modification enhanced thecytokine production in response to autologous tumor (FIG. 47 ),percentage reduction relative to TILs cocultured with autologous digestwithout blocking agents was assessed. The percentage of cytokine releasewas not significantly different between Non-Td and Td TILs in any of theconditions assessed. Antibodies blocking MHCI, MHCII, and both MHCI andMHCII in combination significantly decreased IFN7 release of both Non-Td(32.7%, 43.1%, and 43.1%, respectively) and Td TILs (35.0%, 27.0%, and27.3%, respectively), relative to TIL coculture with autologous digestwith no antibody present. Additionally, inhibition of cytokine releaseby the blocking reagents resulted in reduction that was notsignificantly different between any of the blocking conditions and TILsalone. Isotype control antibody had little effect on IFN7 release fromTd (79.1%) or Non-Td TILs (92.7%), relative to TIL coculture withautologous digest with no antibody present. Reductions in IFN7 releasefrom TIL by MHC blocking antibodies were statistically significant incomparison to isotype control and levels of inhibition were notstatistically significantly different between Td and Non-Td TILsdemonstrating equivalent dependence upon MHC-restricted antigenrecognition.

Assessment of robust TIL effector function and the full potential ofCoStAR to enhance these functions was demonstrated using a range ofengineered cell lines (Table 18). K-562 and BA/F3 cell lines wereengineered to express surface bound OKT3 (to induce a CD3 mediatedsignal 1) or FOLR1 (CoStAR mediated signal 2) or both OKT3 and FOLR1(for signal 1 and 2). Additionally, the ovarian carcinoma cell lineOVCAR-3, which endogenously expresses FOLR1 (63.68% FOLR1+), wasengineered to express surface bound OKT3. Using these cell lines, Non-Tdand Td TIL were cocultured to assess the impact of CoStAR-mediatedeffector functions by evaluating cytokine production and secretion byintracellular flow cytometry and MSD immunoassay, respectively.

TABLE 18 Engineered cell lines Parental line ManufacturerDerived/Engineered line K-562 ATCC K-562 (no signal) K-562-OKT3(signal 1) K-562-FOLR1 (signal 2) K-562-OKT3-FOLR1 (signal 1 & 2)OVCAR-3 ATCC OVCAR-3 (signal 2) OVCAR3-OKT3 (signal 1 & 2) BA/F3 DSMZBA/F3 (no signal) BA/F3-OKT3 (signal 1) BA/F3-FOLR1 (signal 2)BA/F3-OKT3-FOLR1 (signal 1 & 2)

Intracellular flow cytometry was used to enumerate cytokine producingcells after 16-hour coculture with engineered K-562 and OVCAR-3 celllines (FIG. 49 ). IL-2 producing cell frequencies in response to K-562and K-562-FOLR1 were minimal for CD4+(1.47-4.17%) and CD8+(0.23-3.73%)cells across all populations of interest (FIG. 49A). Culture withK-562-OKT3 resulted in IL-2+ cell frequencies of 23.0-34.0% and20.0-39.7% for CD4+ and CD8+ cell populations, respectively. Nostatistically significant difference in IL-2 producing cell frequencieswas observed between Non-Td and anti-FOLR1 CoStAR+/− populations uponcoculture with control cell lines, with the exception of CD8+ anti-FOLR1CoStAR+ Td TILs which were higher than anti-FOLR1 CoStAR− Td TILs, butnot Non-Td TILs (39.7±21.7% vs 20.0±15.1% vs 24.8±13.4%, respectively).Coculture with the K-562-OKT3-FOLR1 cell line resulted in significantlyhigher frequencies of CD4+IL-2 producing anti-FOLR1 CoStAR+ Td TILs witha marked increase of 1.84- and 2.38-fold relative to anti-FOLR1 CoStAR−Td and Non-Td TILs, respectively (54.9±20.5% vs 29.9±19.4% vs23.1±13.4%, respectively). The same observation was true for theCD8+IL-2+ cell frequencies with a 2.53- and 2.29-fold increase inanti-FOLR1 CoStAR+ Td TILs compared to anti-FOLR1 CoStAR− Td and Non-TdTILs, respectively (53.0±23.9 vs 21.0±15.5% vs 23.2±12.2%,respectively).

TILs cultured alone or cocultured with OVCAR-3 were characterized byminimal frequencies of IL-2 producing cells for both CD4+(0.78-1.38%)and CD8+(0.22-0.80%) populations (FIG. 49A). Upon stimulation with bothsignals provided by the OVCAR-3-OKT3 cell line, a significant increasein the frequency of IL-2 producing cells was observed by the anti-FOLR1CoStAR+ Td TILs (39.1±14.5%) in the CD4+ population with a 1.74- and a2.46-fold increase in comparison to anti-FOLR1 CoStAR− Td TILs(22.4±14.0%) and Non-Td TILs (15.9±9.47%), respectively. Similarly, inthe CD8+ population, anti-FOLR1 CoStAR+ Td TILs (34.1±13.2%)demonstrated a 2.65- and 2.36-fold increase in IL-2 producing cellfrequencies compared to anti-FOLR1 CoStAR− Td TILs (12.9±10.2%) andNon-Td TILs (14.5±9.58%), respectively.

CD4+ TNFα positive cell frequencies were significantly higher in theanti-FOLR1 CoStAR+ Td TILs cocultured with K-562-OKT3 compared toanti-FOLR1 CoStAR− Td TILs (75.9±8.35% vs 66.2±5.04%), and withK-562-FOLR1 compared to Non-Td TILs (12.6±7.08% vs 0.73±0.34%) (FIG.49B). No significance was observed in the same conditions for the CD8+TNFα positive cell populations. Stimulation with K-562-OKT3-FOLR1expressing both signals resulted in higher frequencies of TNFα producingCD4+ cells relative to both anti-FOLR1 CoStAR− Td with 1.31-foldincrease and Non-Td TILs with 1.39-fold increase (89.3±6.15% vs68.1±4.99% vs 64.2±14.0%, respectively). The same trend was observed inthe CD8+ population, however, statistical significance was only reachedwhen comparing anti-FOLR1 CoStAR+ Td TILs to anti-FOLR1 CoStAR− Td TILs(1.25-fold increase), but not the Non-Td TILs (1.14-fold increase)(90.2±5.35 vs 72.2±9.92% vs 79.3±6.30%, respectively).

In conditions where TILs were cultured alone, small but significantdifferences in TNFα percentage were observed between the anti-FOLR1CoStAR+ Td TILs compared to anti-FOLR1 CoStAR− Td TILs inCD4+(5.81±3.38% vs 1.85±1.42%) and CD8+(4.85±2.46% vs 1.53±0.76%) cellpopulations (FIG. 49B). There were no significant differences comparedto Non-Td TILs in either population. In the presence of OVCAR-3expressing signal 2, a slight but significantly higher CD4+ TNFα+ cellfrequencies were detected in the anti-FOLR1 CoStAR+ Td TILs relative toboth the anti-FOLR1 CoStAR− Td and Non-Td TILs (8.29±4.34% vs 1.00±0.78%vs 0.40±0.23%, respectively). On the contrary, no differences wereobserved between these populations in the CD8+ cells. Importantly,significantly higher frequencies of CD4+ TNFα producing cells weredetected upon coculture of the TILs with the OVCAR-3-OKT3 cell line inanti-FOLR1 CoStAR+ Td TILs (83.5±6.99%) with a 1.24- and 1.37-foldincrease compared to anti-FOLR1 CoStAR+ Td TILs (67.4±5.35%) and Non-TdTILs (60.9±10.9%), respectively. Similar frequencies were detected inthe CD8+ cell population, with a 1.31-fold increase between anti-FOLR1CoStAR+ Td and anti-FOLR1 CoStAR− Td TILs (88.5±3.72% vs 67.8±10.3%) anda 1.21-fold increase between anti-FOLR1 CoStAR+ Td and Non-Td TILs(88.5±3.72% vs 73.2±4.57%).

Collectively, these results demonstrate a significant increase in thefrequencies of IL-2+ and TNFα+ cells in the CoStAR+ fraction of themodified TILs compared to the CoStAR-fraction and Non-Td TILs whencoculture with engineered cell lines expressing both signals. Althoughminimal, some background of higher frequencies of TNFα+ cell populationswere observed in both CD4+ and CD8+ cells. Similarly, some enhancementof the CoStAR effect was observed by higher frequencies of CD8+IL-2+ andCD4+ TNFα+ cells in cocultures with K-562-OKT3 cell line, which could bepotentially explained by the low levels of FOLR1 expression on theK-562.

In addition to assessing the frequency of activated T cells by flowcytometry, MSD immunoassay was used to evaluate levels of secretedcytokines upon coculture with K-562 and BA/F3 engineered cell lines(Table 18). Cytokine secretion was minimal in cocultures with wild typecell lines or those engineered to express signal 2 alone (FIG. 50 ).IL-2 release stimulated by K-562-OKT3-FOLR1 cell lines was higher in theTd TILs (2682 μg/mL) compared to the Non-Td TILs (521.5 μg/mL)demonstrating a 5.14-fold increase (FIG. 50A). Similarly, in TILscocultured with BA/F3-OKT3-FOLR1, IL-2 secretion was 9.31-fold higher inthe Td TILs compared to Non-Td TILs (FIG. 50A). Significantly higherTNFα production was also detected in Td TILs compared to Non-Td TILswhen cocultured with K-562-OKT3-FOLR1 (2337 μg/ml vs 1211 μg/mL,1.93-fold increase) and BA/F3-OKT3-FOLR1 (900.3 μg/mL vs 278.0 μg/mL,3.24-fold increase).

IL-13 and IFN7 production was also significantly higher upon stimulationwith cell lines expressing both signals (FIG. 50C-D). More specifically,in response to stimulation with K-562-OKT3-FORL1 Td TILs secreted highlevels of IL-13 (1590 μg/mL) compared with Non-Td TILs (587.0 μg/mL),showing a 2.71 fold higher secretion by OKT3-FORL1 Td TILs. Similarly, a2.26-fold higher amount of IFN7 was observed in OKT3-FORL1 Td TILs(194559 μg/mL) vs Non-Td TILs (86183 μg/mL). Similarly, IL-13 and IFN7concentrations were significantly elevated in Td TILs cocultured withBA/F3-OKT3-FOLR1 over Non-Td TILs in the same conditions with a 4.35-(983.9 μg/mL vs 226.1 μg/mL) and 5.13-fold (65594 μg/mL vs 12786 μg/mL)greater cytokine secretion, respectively (FIG. 50C-D). Interestingly, asignificantly higher IFN7 secretion was observed by Td TILs compared toNon-Td TILs in cocultures with K-562-OKT3 (1.33-fold increase, 98804μg/mL vs 74917 μg/mL) which could be attributed to the low levels ofFOLR1 expression on the K-562 cells.

To determine the impact of CoStAR modification on the cytotoxic capacityof TILs, flow cytometry-based and xCELLigence-based killing assays wereconducted against BA/F3 and OVCAR-3 engineered cell lines, respectively(FIG. 51 ). In cocultures assessed by flow cytometry, OKT3 wassufficient to induce cytotoxicity as Non-Td and Td TILs killedspecifically BA/F3 cells expressing either OKT3 or both OKT3 and FOLF1with no significant differences detected (FIG. 51A). Importantly, nocytotoxicity was observed against BA/F3 or BA/F3-FOLR1 demonstrating therequirement of signal 1 to induce killing. Similar observations weretrue for cocultures with OVCAR-3 cells as target cell lines using RTCAassays (FIG. 51B). In cocultures with OVCAR-3 OKT3 expressing bothsignals, there was no significant difference in the AUC (NCI×hours)between Non-Td and Td-TILs at any E:T ratio tested. OVCAR-3 cells alonewere not eliminated at early timepoints, however a slight decrease inNCI of OVCAR-3 cells in 4 of 5 donors occurred following addition ofNon-Td or Td TILs at an E:T 1:5 ratio at later time points.

Collectively, these data demonstrate a lack of cytotoxicity mediated byCoStAR bearing TIL against target cells expressing CoStAR target alone.Engagement of TCR was sufficient in mediating cytotoxicity in bothNon-Td and Td TILs in all assays, showing no impact of CoStAR oncytotoxicity in the presence of a potent signal 1 such as OKT3.

Example 17 Materials and Methods

Co-culture was set up at an effector to target ratio of 8:1 (E:T, 1×105cells: 1.25×104 cells) for the cell lines. T cells were resuspended at1×106 cells/mL in complete TCM and used at 100 μL per well. Cell linetargets were resuspended at 1.25×105 cells/mL in complete TCM and usedat 100 μL per well. T-cells alone and targets alone were set up asnegative controls. T-cells stimulated with PMA (50 ng/mL)/Ionomycin (1g/mL) were set up as positive controls. 100 μL per well was added toTILs alone, targets alone and TILs stimulated with PMA/I. Final volumewas 200 μL per well. Triplicates were set up where possible. Cells wereincubated at 37° C. for 24 hours. 24 hours post incubation plates werespun down at 400×g for 5 minutes. Supernatant was collected anddistributed 60 μL aliquots of each into 3×96 well U-bottom plates.Supernatant was frozen at −80° C., 3 plate aliquots each. V-PLEXProinflammatory Panel 1 Human Kit form MSD was used as per manufacturersinstructions. One aliquot of each sample was thawed to be used. Initialdilution was performed in RPMI medium, and final dilution was performedusing Diluent 2 as per manufacturers instructions

Evaluation of CoStAR expressing cells was conducted on 1e5 live Tcells/well. Cells ere centrifuged (400 g, 5 min), followed by removal ofsupernatant (flicking) and washing cells in 200 uL PBS, centrifugation(400 g, 5 min) and removal of supernatant (flicking). Cells wereresuspended in 100 uL diluted LiveDead Viability dye (1:1000 in PBS) andincubated for 15 min @ RT in the dark. 100 uL PEF was added to cells andcells were centrifuged (400 g, 5 min), supernatant was removed(flicking) and cells were washed in 200 uL PEF, centrifuged (400 g, 5min) and supernatant was removed (flicking). 5) Cells were resuspendedin 100 uL diluted FcR block (1:100 in PEF) and incubated for 15 min @ RTin the dark. 100 uL PEF was added to cells and cells were centrifuged(400 g, 5 min), supernatant was removed (flicking) and cells were washedin 200 uL PEF, centrifuged (400 g, 5 min) and supernatant was removed(flicking). Cells were resuspended in 100 uL diluted antibody mastermixand incubated for 25 min at 4° C. in the dark. Cells were centrifuged(400 g, 5 min), supernatant was removed (flicking) and cells were washedin 200 uL PEF, centrifuged (400 g, 5 min) and supernatant was removed(flicking). 100 uL PEF was added to cells and cells were centrifuged(400 g, 5 min), supernatant was removed (flicking) and cells were washedin 200 uL PEF, centrifuged (400 g, 5 min) and supernatant was removed(flicking). 100 uL PEF was added to cells and flow cytometry resultswere acquired.

Results

To evaluate the relevance of MSLN as a CoStAR target, mSLN expressionwas evaluated in various cancer cell types including both primary andmetastatic tumors. The results from the evaluation are shown in FIG.61A-B, where the % of mesothelin+ cells approached 100% in multiplesamples and all but four cancer types tested (cervical SSC, gastricadenocarcinoma, NSCLC SSC, NSCLC NOS) showed replicates with at least50% of mesothelin+ cells.

Accordingly, CoStARs bearing anti-MSLN scFvs were developed. Sixdifferent CoStARs comprising the same linker, spacer, TM, andintracellular domains but possessing different scFvs directed againstMSLN are depicted in FIG. 62 . Expression of the CoStARs in T cells wasevaluated by measuring markers for MSLN-PE and CD34. FIG. 62demonstrates high expression of both CD34 and MSLN-PE regardless ofwhich scFv was included in the CoStAR. Flow cytometry plots of CD34 andMSLN-PE expression demonstrate percentages T cells double positive forCD34 and rhMSLN-PE ranging from 12.87-46.59% (See FIG. 63A-B). CoStARexpression in transduced T cells was further examined by evaluatingexpression of MSLN-PE and CD34 further gated for CD4 and CD8 T cells(See FIG. 64 ). As can be seen in FIG. 64 , CD4 expression levels arecomparable to those seen in CD3, whereas CD34 expression is slightlydecreased in CD8 cells for all scFvs. Alternatively, transduced cellswere placed into a rapid expansion protocol (REP) for 14 days followingCD34 sorting (See FIG. 65 ). Similarly to what was observed in FIG. 64 ,CD4 expression levels were comparable to those seen in CD3, however,CD34 expression levels were higher in CD8 cells than was previously seenwithout the REP (See FIG. 65 ). Data from a separate experiment alsoinvolving MSLN CoStARs expanded with a REP is present in in FIG. 66 ,showing high expression levels of CD34 and MSLN-PE across threebiological replicates.

To evaluate function of the MSLN targeting CoStARs, donor PBMCs weretransduced with a lentivirus for 1 of the 6 MSLN targeting CoStARs.Transduced cells were allowed outgrowth, followed by CD34 selection anda 12 day REP, followed by co-culture with the naturally MSLN and OKT3expressing Ovcar3 cell line, where cytokine release by CoStAR expressingcells was evaluated (See FIG. 67 ). MSLN expression was evaluated inOvcar3 and Ovcar3-OKT-3 cells via flow cytometry (See FIG. 68 ). FIG. 66demonstrates that both Ovcar3 and Ovcar3-OKT-3 cells have similar levelsof MSLN expression at 37.49 and 36.6% of cells positive for MSLNrespectively.

Cytokine expression in healthy donor T cells transduced with scFVanti-MSLN CoStARs expressing varied scFv domains and either a CD8 spaceror truncated CD28 spacer and cocultured with K562 cell lines wasassessed when CoStAR expressing cells were cocultured with Ovcar andOvcar-OKT3 cells (FIG. 69 ). FIG. 69 shows that optimal expression ofIL-2, IFNγ, and TNFα required both signal 1 and signal 2 as evidenced bythe robust responses in Ovcar3-OKT-3 cells and weak responses in Ovcar3cells. Furthermore, CoStARs with both CD8 spacers or truncated CD28spacers were both capable of robust cytokine responses (See FIG. 69 ).

A similar experiment was performed in FIG. 70 where healthy donor Tcells were transduced with CoStARs with varied scFvs and CD28, truncatedCD28, or CD28 spacer domains. The CoStAR expressing cells wereco-incubated with K562-MSLN or K562-MSLN-OKT-3 cells and cytokinerelease was evaluated. FIG. 70 shows that optimal expression of IL-2,IFNγ, and TNFα by the CoStAR expressing cells required both signal 1 andsignal 2 as evidenced by the robust responses in K562-MSLN-OKT-3 cellsand weak responses in K562-MSLN cells. Additionally, CoStAR expressingcells with CD28 spacers exhibited slightly higher secretion of IL-2 andIFN7 in comparison to the truncated CD28 and CD8 domains (FIG. 70 ).

Example 18 Materials and Methods

Tumor digests were thawed in TCM base media supplemented with 10% FBS,1× Gentamycin/Amphotericin (500× stock), 50 ug/mL vancomycin and 3000IU/mL IL2. The cells were resuspended at a concentration of 0.5 or 1×10⁶cells/mL for seeding. For each sample, cells were seeded at either0.5M/mL in a 24 well plate or 1M/2 mL in 6 well plate. Cells were seededsuch that one well was kept for non-Td TIL and another well for anti-CEACoStAR-Td TIL generation. Cells were placed in a humidified incubator37° C. with 5% CO2.On day 3 and 4, the appropriate volume of lentiviruswas diluted in 0.1 mL media for 24 well plate or 0.5 mL media for 6 wellplate and added to the wells to be transduced. On day 8, cells were fedwith TCM base media supplemented with 10% FBS, 1×Gentamycin/Amphotericin (500× stock), 50 ug/mL vancomycin and 6000 IU/mLIL2. On day 10, cells were collected from all conditions and countedusing Vicell. Based on cell counts, TILs were stimulated in a 6 wellGRex with OKT3 (30 ng/mL), IL2 (3000 U/mL) and irradiated feeders at a1:200 ratio in TCM base media with 8% human AB serum. On day 15, thecells were transferred to a GRex 6M and TCM base media with 8% human ABserum and IL2 at 3000 U/mL was added. On day 18, TILs were counted andeither media was changed for cell counts <1e6 cells/mL or conditionswere split of cell counts >1e6 cells/mL. All conditions were harvestedand frozen on day 21.

Results

The present example tests for fold expansion of T cells from fourdifferent tumor types following transduction with either CTP386.1 orCTP387.1 CEA targeting CoStAR constructs. FIG. 71 shows variable levelsof expansion across T cells from different tumor types, with cells fromCRC generally having less expansion than cells from NSCLC, OV, and MELtumors.

In addition to expansion, transduction efficiency of the two constructswas also evaluated and the results are shown in FIG. 72 . As shown inFIG. 72 , transduction efficiency ranged from approximately 20-55% withCRC11974 having the highest transduction efficiency for both constructsand CRC11959 having the lowest.

Transduction efficiency was also broken down across T cell subtypes. CD4and CD8 T cells were evaluated for CoStAR transduction efficiency andthe results are shown in FIG. 73 . As can be seen in FIG. 73 ,transduction efficiency was higher in CD4 cells for CRC and ovariantumor derived T cells, while transduction efficiency was higher in CD8 Tcells for NCSLC and melanoma derived T cells.

In addition, T cell memory phenotype was evaluated across T cellstransduced with the 2 CoStAR constructs. Expression levels of CCR7 andCD45RA were evaluated on day 21 to determine T cell memory phenotype andthe results are shown in FIG. 74 .

Example 19 Materials and Methods

Effector (ie, Non-Td and Td) T cells were rested at 1×10⁶ cells/mL inTCM (T cell media) and incubated overnight at 37° C. with 5% CO2. On theday of coculture, the effector T cells were collected and resuspended at1×10⁶ cells/mL. Target cell lines (ie, K562 WT, K562 OKT3, K562 CEACAM5and K562 OKT3 CEACAM5) were collected and resuspended at 1×10⁵ cells/mL.50 uL T cells and targets were plated in a 96 well U bottom plate toachieve a 10:1 E:T ratio. The volume of media in all wells were made upto 200 uL. Following overnight coculture, plates were collected andcentrifuged at 400×g for 5 minutes. 100 mL of supernatant was collectedfrom each well and stored at −80° C. prior to analysis of cytokinecontent using an MSD V-Plex Plus Proinflammatory Panel 1 kit. The assaywas carried out according to the manufacturer's instructions andanalysis performed using MSD discovery workbench software.

Results

Sorted transduced TILs underwent functional testing for cytokine releasefollowing co-culture with K562 target cells expressing signal 1, signal2, or both signal 1 and signal 2. The experiment was carried out andlevels of IL-2, TNFα, and IFN7 were measured the results are shown inFIGS. 75-78 . FIG. 75 shows the results for CRC cells, FIG. 76 showsNSCLC, FIG. 77 shows OV-9962, and FIG. 78 shows melanoma derived Tcells. In all TIL sources, target cells expressing both signal 1 andsignal 2 results in higher cytokine levels than target cells with signal1 or 2 alone. Additionally, IFN7 was induced at higher levels than IL-2or TNFα (See FIGS. 75-78 ).

Example 20 Materials and Methods

Effector (ie, Non-Td and Td) T cells were rested at 1×10⁶ cells/mL inTCM (T cell media) and incubated overnight at 37° C. with 5% CO2. On theday of coculture, the effector T cells were collected and resuspended at1×10⁶ cells/mL. Irradiated target cell line (ie, K562 OKT3 CEACAM5) wascollected and resuspended at 0.2×10⁶ cells/mL. 50 uL T cells and targetswere plated in a 96 well U bottom plate to achieve a 5:1 E:T ratio. Thevolume of media in all wells were made up to 200 uL. Plates wereincubated 37° C. with 5% CO₂ and re-stimulated with targets weekly.

To re-stimulate with targets every week (ie, day 7, 14, 21, 28 and 35),20 uL cell suspension was collected from each well to obtain cell countsusing a Vicell and assess fold expansion. 50 uL cell suspension wascollected to stain and evaluate CoStAR expression levels. Remaining cellsuspension was centrifuged at 400×g for 5 mins and supernatantdiscarded. The cell pellets were resuspended in 50 uL media. IrradiatedK562 OKT3 CEACAM5 cell line was resuspended at 1×10⁶ cells/mL. Targetswere then added at 5:1 ratio to each well based on effector cell countsobtained. The volume of media in all wells were made up to 200 uL andincubated for a week at 37° C. with 5% CO2 until the next round ofre-stimulation.

Results

Fold expansion and transduction efficiency were evaluated in CoStARtransduced cells following weekly stimulation with K562 OKT3 CECAM5target cells. The evaluation was conducted and the results are shown inFIGS. 79-82 . FIG. 75 shows the results for CRC 11959 and CRC 11974cells, FIG. 76 shows NSCLC 9332 and NSCLC 9596, FIG. 77 shows OV-9962,and FIG. 78 shows MEL CC50, MEL 11909, and MEL 17614 derived T cells. Ascan be seen in FIG. 79-82 , CoStAR expressing cells show enhancedproliferation compared to nontransduced cell co-incubated with thetarget cells, particularly the cells transduced with CTP387.1 construct.Transduction efficiency on day 21 or 28 ranged from approximately20-80%, with the lowest transduction efficiency in OV9662 cells and thehigh levels for both constructs seen in MEL CC50 and MEL 17614.

Example 21 Materials and Methods

The following examples provide the details regarding Example 17 above,the results of which are presented in FIGS. 70A and 70B. Cytokineexpression analysis was performed for HD T cells transduced with CEACAMor MSLN ScFv co-cultured with CEACAM and MSLN K562 cell lines. The firstobjective of this work was to assess if there is an enhancement ofcytokine expression (T-cell function) when co-culturing T-cellstransduced with anti-CEA scFv candidates with either CEACAM5 (signal 2only) or CEACAM5/OKT3 (signal 1+2) expressing K562.luc.puro cell lines.IL-2, IFN-γ or TNF-α levels were measured using MSD for each of the scFvconstructs (MFE23, MFE23 (Q>K), hMFE23, CEA6, BW431/26 and hT84,66)transduced T-cells. K562.luc.CEACAM5 and K562.luc.puro.OKT3.GFP.CEACAM5were utilized as they should provide a cleaner system then using theLovo cell line (CEACAM5+). The second objective was to assess if thereis an enhancement of cytokine expression (T-cell function) whenco-culturing T-cells transduced with anti-MSLN scFv candidates witheither MSLN (signal 2 only) or MSLN/OKT3 (signal 1+2) expressingK562.luc.puro cell lines. IL-2, IFN-γ or TNF-α levels were measuredusing MSD for each of the scFv constructs (MFE23, MFE23 (Q>K), hMFE23,CEA6, BW431/26 and hT84,66) transduced T-cells. K562.luc.CEACAM5 andK562.luc.puro.OKT3.GFP.CEACAM5 were utilized as they should provide acleaner system then using the Ovcar3 cell line (CEACAM5+). This workincludes the Production of HD with CoStAR2 production of HD with CoStAR2CEA ScFv and MSLN ScFv constructs to assess expression andfunctionality. The co-culture was set up at an effector to target ratioof 8:1 (E:T, 1×105 cells:1.25×104 cells) for the cell lines. Allco-cultures were incubated for 24 hours before collection and freezingof the supernatant.

The following is the entire process of this experiment in regards to theorder of the work that was completed: 1. PBMC isolation from buffy coatsby density gradient separation using Ficoll-Paque centrifugation. 2.T-cell isolation using the Human T-cell isolation kit (STEMCELL). 3.Transduction of T-cells. 4. Transduction assessment by flow cytometrystain. 5. Day 5 count of cells. 6. CD34 selection of cells usingMicroBeads (Milteni Biotec). 7. Transduction assessment by flowcytometry pre-REP. 8. Rapid Expansion Protocol of T-cells (REP). 9.Transduction assessment by flow cytometry post-REP. 10. Post-REP cellcounts. 11. Co-culture set up with antigen or OKT3 presenting engineeredlines (K562.luc.puro.CEACAM5, K562.luc.puro.OKT3.GFP.CEACAM5,K562.luc.puro.MSLN or K562.luc.puro.OKT3.GFP.MSLN). 12. Analysis ofsupernatant from co-culture using the Meso Scale Discovery (MSD)platform (to measure IFN-γ, IL-2 & TNF-α secretion).

Media Preparation

PEF media was prepared by adding 2 mL of EDTA to 500 mL phosphatebuffered saline (PBS) bottle, followed by adding 2.5 mL fetal bovineserum. Complete TCM was prepared by adding 50 mL fetal bovine serum to450 mL bottle of RPMI 1640, followed by adding 5 mLpenicillin-streptomycin, 5 mL HEPES solution, and 500 μL2-Mercaptoethanol (50 mM).

Preparation of the Cells Before T Cell Isolation

NBC PBMC were obtained from processed buffy coats in 210831 4× HD PBMCisolation and banking. 5× vials of each donor PBMCs (NBC571, NBC572,NBC573) were thawed in a 37° C. water bath. To minimise the time thecells spend thawed in the freezing media, vials were thawed and washedin batches of 5 vials. The contents of the vials were transferred intoone 50 mL falcon tube per donor and top up with TCM. The cells werecentrifuged at 400×g for 5 minutes and washed once with 50 mL TCM each.The cells were counted using the Vicell-Blu (10 μL of sample+190 μLDPBS, Dilution 1:20)

T-Cell Isolation

The PBMCs were washed in PEF and then resuspended at 5×107 cells/mL. 2mL of the resuspended cells were transferred per 5 mL facs tubes. Tcells were isolated using EasySep™ Human T Cell Isolation Kit accordingto the manufacturer's protocol. After the isolation, cells werecollected, transferred the into 2×50 mL falcon tubes and washed oncewith complete TCM. Cells were resuspended in 20 mL of complete TCM per50 mL falcon. 10 μL of each were taken and resuspend in 190 μL of PBS(Dilution 1:20). The cells were counted using the ViCELL-Blu.

T-Cell Activation by DynaBeads

The cells were collected from the isolations and transferred onto 50 mLfalcon tubes. The isolated T cells were centrifuged at 400×g for 5minutes. The T cells were washed in complete TCM and then resuspended at1×106 cells/mL based on the total counts. The T cells were transferredinto T75 flasks. 200 IU/mL IL-2 (1:5000 dilution of the 106 IU/mL stock)were added. The CTS Dynabeads™ CD3/CD28 magnetic beads were resuspendedin the vial (i.e., vortex for 30 sec). 0.83 μL CTS Dynabeads™ CD3/CD28were added per ml of T-cell suspension (1:3 bead:cell ratio). Theculture was gently rocked to resuspend the Dynabeads and incubated for48 hours.

Preparation of T Cells for Lentiviral Transduction

The activated T cells were transferred from the flasks into a 50 mLfalcon tube. And centrifuged at 400×g for 5 minutes. The cells wereresuspended in 20 mL of complete T cells each. 10 μL of each were takenand resuspended in 190 μL of PBS (Dilution 1:20). The cells were countedusing the ViCELL-Blu. The cells were centrifuged at 400×g for 5 minutesand resuspend at 1×106 cells/mL. The appropriate number of cells andvirus needed was estimated—for 10⁵ transduced cells you will need 100 μLof lentivirus of chosen titre.

Lentiviral aliquots were thawed at 37° C. water bath. The amount of timethe virus is at room temperature was minimized by placing in the 4° C.fridge. The number of T cells needed per condition was transferred intoa sterile Eppendorf tube (i.e., 5×105 T cells) and spun it down (400×g,5 minutes, RT). Supernatant was discarded, excess media was removed withpipette and resuspended in lentivirus at a MOI of 5 for all eg. 100 μLper 105 T-cells (i.e., 5×105 T cells in 500 μL of lentivirus). 0.4 mg/mLpolybrene was prepared by diluting the stock (100 mg/mL) 25× withcomplete TCM (to get 4 mg/mL). 0.1 μL of 0.4 mg/mL polybrene was addedper 100 μL (i.e., 0.5 μL into 500 μL of virus). 104 IU/mL IL-2 wasprepared by diluting the stock (106 IU/mL) 100× with complete TCM. 2 μLof 104 IU/mL IL-2 was added per 100 μL (i.e. 10 uL into 500 uL ofvirus). 500 μL of cells was properly resuspended and plated with virusper well onto a flat-bottom 48-well plate. The plate was spun at 1200×gfor 1.5 hour at 32° C. 8. The cells rested after spin for 5 hours. Atthe end of incubation top up to 700 μL/well with complete TCM with 200IU/mL IL-2. 10. Incubation proceeded for 72 hours.

Removal of Dynabeads from Culture

The cells were pipetted up and down thoroughly (to detach Dynabeads) andtransferred into an Eppendorf tube/FACS tube. Ensure this is doneeffectively for maximum recovery of cells. The tube was pressed againstthe small magnet (Eppendorf tube)/place it into the big magnet (FACStube) for ˜3 min. The cells were carefully transferred (Eppendorf)/pourthem out (FACS), onto a new falcon tubes and centrifuged at 400×g for 5minutes. The supernatant was discarded and resuspend the cells in 1.2 mLeach. The cells were counted using the ViCELL-Blu 1. 10 μL of each weretaken and resuspended in 190 μL of PBS (Dilution 1:20).

The cells were resuspended at a concentration of 1×10⁶ cells/mL. 200IU/mL IL-2 were added (1:5000 dilution of the 10⁶ IU/mL stock). 2.Before adding IL-2, 1×10⁵ of each condition were taken for transductionassessment. Cell should be kept in complete TCM with 200 IU/mL IL-2 andwill grow stably for at least 2 weeks (donor variability). The next dayand onwards, cells can be stained for transduction efficiency, sortedand expanded, or used for functional assays.

Preparation of Cells for Stain

The NBC571, NBC572 and NBC573 T cells (MOCK and transduced) werestained. 1×10⁵ cells per well were used for the staining. 1×10⁵ cellsper well were placed in a 96-well round-bottom plate. The well contentswere clearly labelled with an appropriate identifier using an alcoholresistant marker pen. IL-2 (200 IU/mL) was added to the rest of thecells and the cells were placed in a 37° C. incubator 5% CO2.

Staining Protocol

The wells were topped up to 200 μL using PBS and the plate was spun at500×g for 3 minutes. The supernatant was discarded by flicking off theplate. Fixable Viability Dye Efluor™450 was prepared by diluting stock1:1000 in PBS. 100 μL of Viability Dye preparation is required persample. 100 μL of viability stain per sample was added and samples wereincubated at RT for 10 minutes in the dark. After the incubation period,the wells were topped up with 100 L of BD stain buffer and the plate wasspun at 500×g for 3 minutes. The supernatant was discarded by flickingoff. A working solution of Fc blocking reagent was prepared by dilutingstock 1 in 100 in BD stain buffer. 100 μL of Fc blocking solution isrequired per sample. 10. 100 L of the Fc blocking solution was added persample and incubated in RT for 10 minutes in the dark. After theincubation period, the wells were topped up with 100 μL of stain bufferand the plate was spun at 500×g for 3 minutes. The supernatant wasdiscarded by flicking off. 13. The CEA-Fc protein was prepared bydiluting in BD stain buffer. 100 μL of the CEACAM5 protein FC/BD wasmixed to the appropriate wells. For wells not receiving CEACAM5protein-FC mix 100 μl of BD stain buffer alone was added. The plate wasincubated at 4° C. for 25 minutes in the dark (cover plate with foil).After the incubation period, the wells were topped up with 100 μL of BDstain buffer and spun at 500×g for 3 minutes. The cells were washed 2×with 200 μL of BD stain buffer. Extracellular staining mastermix and FMOcontrols were prepared by diluting antibodies in BD stain buffer. 100 ulof the staining mix antibodies and the conjugated antibodies were added.The plate was incubated at 4° C. for 25 minutes in the dark (cover platewith foil). After the incubation period, the wells were topped up with100 μL of BD stain buffer and spun at 500×g for 3 minutes. Thesupernatant was discarded by flicking off. The plate was washed 2× with200 μL of BD stain buffer. After the second wash 100 μL of BD stainbuffer was added. Signal was acquired on the NovoCyte. Files were savedand analyzed.

Example 22 CD34 Miltenyi MicroBeads Enrichment

Each donor was processed individually. The cells were collected and cellsuspension was centrifuged at 400×g for 5 minutes. The cells wereresuspended in 1 mL of complete TCM and transferred 10 μL each to 96well U-bottom plate for counts. 190 μL of PBS was added (1:20 dilutionof sample). Cells were counted using ViCELL-Blu.

The cells were topped up with 9 mL of cold PEF and centrifuged at 400×gfor 5 minutes. Supernatant was aspirated completely. Cells were washedonce with cold PEF buffer and resuspended in a final volume of 300 μL ofcold PEF buffer for each (MOCK cells were not counted rested in IL-2until REP).

Magnetic Labelling

50 μL of FcR Blocking Reagent was added for up to 10⁸ total cells. 50 μLof CD34 MicroBeads were added for up to 10⁸ total cells. 3. Mix wasmixed well and incubated for 30 minutes in the refrigerator (2-8° C.).Cells were washed by adding 5 mL of buffer for up to 10⁸ cells andcentrifuged at 400×g for 5 minutes. Supernatant was aspiratedcompletely. Up to 10⁸ cells were resuspended in 500 μL of cold PEFbuffer.

Magnetic Separation with MS Columns

MS column was placed in the magnetic field of the OctoMACS Separator.Column was prepared by rinsing with 500 μL of cold PEF buffer. Cellsuspension was applied onto the column. Flow-through containingunlabelled cells was collected. Column was washed with 3×500 μL of coldPEF buffer. Unlabelled cells that pass through were collected andcombined with the flow-through from step 3. Washing steps were performedby adding cold PEF buffer (4° C.) aliquots only when the columnreservoir is empty. Column was removed from the separator and placed ona suitable pre-labelled collection tube. 1 mL of cold PEF buffer waspipetted onto the column. The magnetically labelled cells wereimmediately flushed out by firmly pushing the plunger into the column.(Optional) To increase the purity of CD34+ cells, the eluted fractioncan be enriched over a second MS or LS Column. The magnetic separationprocedure was repeated as described above by using a new column.Centrifuge at 400×g for 5 minutes. The supernatant was aspiratedcompletely. The positive fraction was resuspended in 1 mL of completepre-warmed TCM. Note: Take 10 μL of the stain cells pre-sort of each,100 μL of the negative fraction and 20 μL of the positive fractionresuspended in TCM and stain cells in BD stain buffer with CD34− PE (0.5μL/100 μL reaction) beads for 15 minutes at 4° C. After the incubation,100 μL of BD stain buffer was used to top up and mix was centrifuged at500×g for 3 minutes. The cells were resuspended in 100 μL of BD stainbuffer with DRAQ7 (1:200 dilution) and 120 μL of each were acquired onthe Novocyte 3005 to determine selection efficiency. Also, 20 μL each ofthe positive fractions was transferred to a 96 well U-bottom plate forcounts. 180 μL of PBS (1:10 dilution of sample) was added. Cells werecounted using ViCELL-Blu.

Example 23 Rapid Expansion Protocol Set Up Using the R+D Protocol (D12Post Activation) Materials and Methods

The rested MOCK or CD34 selected cells were counted using ViCELL-Blu (10μL of each sample with 190 μL of PBS, 1:20 dilution) and cells wereresuspended at a 1e6 cells/mL. Irradiated feeders were used. The cellswere resuspended at a concentration of 1×10⁸ cells/mL. T cells wereresuspended in complete REP TIL TCM media with IL-2 and OKT3 cells at adensity of 1×10⁶/mL then transfer the to a 6-well G-REX wells. The G-Rexplate was placed in a humidified incubator 37° C.+5% CO2.

Every 2-3 days IL-2 was added to the wells. No further OKT3 was addedafter the first day Day 2 REP (15 days post activation) Added IL-2 2001IU/mL to all REP cells. Day 4 REP (17 days post activation) IL-2 200IU/mL was added to all REP cells. Day 6 REP (19 days post activation) Onday 6, media was removed from each well. For 6-well G-REX 20 mL wasremoved from each well being careful not to disrupt the cells at thebottom of the well and replaced with fresh T-cell media. If the colourof the media changed to yellow before day 6 the media was refreshedearlier. After day 6 if the media colour did change to yellow, the mediawas refreshed again not forgetting to supplement with IL-2 200 IU/mL. Ifthe pH of the media did not show any colour changes the media did notneed to be changed again until the end of the rep. Day 8 REP 10 mL ofmedia was removed from each well and 10 mL of Fresh media was added plusIL-2 (200 IU/mL). Day 11 REP IL-2 (200 IU/mL) was added. Day 14 REP (Day26 post activation) Cells were collected and transferred to 50 mL falcontubes. Cells were centrifuged at 400×g for 5 minutes. Cells wereresuspended in 50 mL of TCM. 4. The cells were counted using ViCELL-Blu(10 μL of sample with 190 μL of PBS, 1:20 dilution). 1×10⁵ cells werestained to assess phenotype and transduction. Remaining cells werefrozen down to be used for co-culture. Cells frozen down to mimicprevious process. Cells to be thawed and rested prior to co-culture withantigen presenting K562.luc.puro CEACAM5 or K562.luc.puro MSLN lines.

Example 24

Co-Culture with Cell Lines for Cytokine Secretion Analysis

Materials and Methods

For the co-culture an effector to target ratio of 8:1 (E:T, 1×10cells:1.25×10⁴ cells) was set up for the cell lines. T cells wereresuspended at 1×10⁶ cells/mL in complete TCM and 100 L was used perwell. For cell line targets, resuspended at 1.25×105 cells/mL incomplete TCM and used 100 μL per well. T cells alone and targets alonewere set up as negative controls. T cells stimulated with PMA (50ng/mL)/Ionomycin (1 μg/mL) were set up as positive controls. T cellsalone were topped up, targets alone and T cells were stimulated withPMA/I, with 100 μL per well. Final volume was 200 μL per well set up astriplicate. 7. The cells were incubated at 37° C. for 24 hours. 24 hourspost incubation the plates were spun down at 400×g for 5 minutes. Thesupernatant was collected and distributed 80 μL aliquots of each into2×96 well U-bottom plates. Supernatant was frozen at −80° C., 2 platealiquots each.

Having thus described in detail preferred embodiments of the presentinvention, it is to be understood that the invention defined by theabove paragraphs is not to be limited to particular details set forth inthe above description as many apparent variations thereof are possiblewithout departing from the spirit or scope of the present invention.

1-43. (canceled)
 44. A chimeric costimulatory antigen receptor (CoStAR)which comprises: an extracellular binding domain that binds tomesothelin (MSLN); a transmembrane domain; a CD28 signaling domain,wherein the CD28 signaling domain comprises the amino acid sequence ofSEQ ID NO: 25; and a CD40 signaling domain, wherein the CD40 signalingdomain comprises the amino acid sequence of SEQ ID NO: 32, wherein theextracellular binding domain comprises any one of: (a) a HCDR1 that isan HCDR1 in SEQ ID NO: 186, a HCDR2 that is an HCDR2 in SEQ ID NO: 186,a HCDR3 that is an HCDR3 in SEQ ID NO: 186, a LCDR1 that is an LCDR1 inSEQ ID NO: 186, a LCDR2 that is an LCDR2 in SEQ ID NO: 186, and LCDR3that is an LCDR3 in SEQ ID NO: 186; (b) a HCDR1 that is an HCDR1 in SEQID NO: 187, a HCDR2 that is an HCDR2 in SEQ ID NO: 187, a HCDR3 that isan HCDR3 in SEQ ID NO: 187, a LCDR1 that is an LCDR1 in SEQ ID NO: 187,a LCDR2 that is an LCDR2 in SEQ ID NO: 187, and LCDR3 that is an LCDR3in SEQ ID NO: 187; (c) a HCDR1 that is an HCDR1 in SEQ ID NO: 188, aHCDR2 that is an HCDR2 in SEQ ID NO: 188, a HCDR3 that is an HCDR3 inSEQ ID NO: 188, a LCDR1 that is an LCDR1 in SEQ ID NO: 188, a LCDR2 thatis an LCDR2 in SEQ ID NO: 188, and LCDR3 that is an LCDR3 in SEQ ID NO:188; (d) a HCDR1 that is an HCDR1 in SEQ ID NO: 189, a HCDR2 that is anHCDR2 in SEQ ID NO: 189, a HCDR3 that is an HCDR3 in SEQ ID NO: 189, aLCDR1 that is an LCDR1 in SEQ ID NO: 189, a LCDR2 that is an LCDR2 inSEQ ID NO: 189, and LCDR3 that is an LCDR3 in SEQ ID NO: 189; (e) aHCDR1 that is an HCDR1 in SEQ ID NO: 190, a HCDR2 that is an HCDR2 inSEQ ID NO: 190, a HCDR3 that is an HCDR3 in SEQ ID NO: 190, a LCDR1 thatis an LCDR1 in SEQ ID NO: 190, a LCDR2 that is an LCDR2 in SEQ ID NO:190, and LCDR3 that is an LCDR3 in SEQ ID NO: 190; (f) a HCDR1 that isan HCDR1 in SEQ ID NO: 191, a HCDR2 that is an HCDR2 in SEQ ID NO: 191,a HCDR3 that is an HCDR3 in SEQ ID NO: 191, a LCDR1 that is an LCDR1 inSEQ ID NO: 191, a LCDR2 that is an LCDR2 in SEQ ID NO: 191, and LCDR3that is an LCDR3 in SEQ ID NO: 191; (g) a VH that is a VH in SEQ ID NO:186, and a VL that is a VL in SEQ ID NO: 186; (h) a VH that is a VH inSEQ ID NO: 187, and a VL that is a VL in SEQ ID NO: 187; (i) a VH thatis a VH in SEQ ID NO: 188, and a VL that is a VL in SEQ ID NO: 188; (j)a VH that is a VH in SEQ ID NO: 189, and a VL that is a VL in SEQ ID NO:189; (k) a VH that is a VH in SEQ ID NO: 190, and a VL that is a VL inSEQ ID NO: 190; or (l) a VH that is a VH in SEQ ID NO: 191, and a VLthat is a VL in SEQ ID NO:
 191. 45. The CoStAR of claim 44, whichfurther comprises an N-terminal signal peptide.
 46. The CoStAR of claim45, wherein the N-terminal signal peptide comprises the signal peptideof h-oncostatin M (OSM).
 47. The CoStAR of claim 44, wherein the CoStARcomprises the amino acid sequence of SEQ ID NO:
 19. 48. The CoStAR ofclaim 44, wherein the CoStAR further comprises a linker, wherein thelinker comprises the amino acid sequence of SEQ ID NO:
 18. 49. TheCoStAR of claim 44, wherein the extracellular binding domain comprises aHCDR1 that is an HCDR1 in SEQ ID NO: 186, a HCDR2 that is an HCDR2 inSEQ ID NO: 186, a HCDR3 that is an HCDR3 in SEQ ID NO: 186, a LCDR1 thatis an LCDR1 in SEQ ID NO: 186, a LCDR2 that is an LCDR2 in SEQ ID NO:186, and LCDR3 that is an LCDR3 in SEQ ID NO:
 186. 50. The CoStAR ofclaim 44, wherein the extracellular binding domain comprises a HCDR1that is an HCDR1 in SEQ ID NO: 187, a HCDR2 that is an HCDR2 in SEQ IDNO: 187, a HCDR3 that is an HCDR3 in SEQ ID NO: 187, a LCDR1 that is anLCDR1 in SEQ ID NO: 187, a LCDR2 that is an LCDR2 in SEQ ID NO: 187, andLCDR3 that is an LCDR3 in SEQ ID NO:
 187. 51. The CoStAR of claim 44,wherein the extracellular binding domain comprises a HCDR1 that is anHCDR1 in SEQ ID NO: 188, a HCDR2 that is an HCDR2 in SEQ ID NO: 188, aHCDR3 that is an HCDR3 in SEQ ID NO: 188, a LCDR1 that is an LCDR1 inSEQ ID NO: 188, a LCDR2 that is an LCDR2 in SEQ ID NO: 188, and LCDR3that is an LCDR3 in SEQ ID NO:
 188. 52. The CoStAR of claim 44, whereinthe extracellular binding domain comprises a HCDR1 that is an HCDR1 inSEQ ID NO: 189, a HCDR2 that is an HCDR2 in SEQ ID NO: 189, a HCDR3 thatis an HCDR3 in SEQ ID NO: 189, a LCDR1 that is an LCDR1 in SEQ ID NO:189, a LCDR2 that is an LCDR2 in SEQ ID NO: 189, and LCDR3 that is anLCDR3 in SEQ ID NO:
 189. 53. The CoStAR of claim 44, wherein theextracellular binding domain comprises a HCDR1 that is an HCDR1 in SEQID NO: 190, a HCDR2 that is an HCDR2 in SEQ ID NO: 190, a HCDR3 that isan HCDR3 in SEQ ID NO: 190, a LCDR1 that is an LCDR1 in SEQ ID NO: 190,a LCDR2 that is an LCDR2 in SEQ ID NO: 190, and LCDR3 that is an LCDR3in SEQ ID NO:
 190. 54. The CoStAR of claim 44, wherein the extracellularbinding domain comprises a HCDR1 that is an HCDR1 in SEQ ID NO: 191, aHCDR2 that is an HCDR2 in SEQ ID NO: 191, a HCDR3 that is an HCDR3 inSEQ ID NO: 191, a LCDR1 that is an LCDR1 in SEQ ID NO: 191, a LCDR2 thatis an LCDR2 in SEQ ID NO: 191, and LCDR3 that is an LCDR3 in SEQ ID NO:191.
 55. The CoStAR of claim 44, wherein the extracellular bindingdomain comprises a VH that is a VH in SEQ ID NO: 186, and a VL that is aVL in SEQ ID NO:
 186. 56. The CoStAR of claim 44, wherein theextracellular binding domain comprises a VH that is a VH in SEQ ID NO:187, and a VL that is a VL in SEQ ID NO:
 187. 57. The CoStAR of claim44, wherein the extracellular binding domain comprises a VH that is a VHin SEQ ID NO: 188, and a VL that is a VL in SEQ ID NO:
 188. 58. TheCoStAR of claim 44, wherein the extracellular binding domain comprises aVH that is a VH in SEQ ID NO: 189, and a VL that is a VL in SEQ ID NO:189.
 59. The CoStAR of claim 44, wherein the extracellular bindingdomain comprises a VH that is a VH in SEQ ID NO: 190, and a VL that is aVL in SEQ ID NO:
 190. 60. The CoStAR of claim 44, wherein theextracellular binding domain comprises a VH that is a VH in SEQ ID NO:191, and a VL that is a VL in SEQ ID NO:
 191. 61. The CoStAR of claim44, wherein the CoStAR comprises the amino acid sequence of SEQ ID NO:192, lacking the signal peptide of SEQ ID NO:
 1. 62. The CoStAR of claim44, wherein the CoStAR comprises the amino acid sequence of SEQ ID NO:210, lacking the signal peptide of SEQ ID NO:
 1. 63. The CoStAR of claim44, wherein the CoStAR comprises the amino acid sequence of SEQ ID NO:228, lacking the signal peptide of SEQ ID NO:
 1. 64. The CoStAR of claim44, wherein the CoStAR comprises the amino acid sequence of SEQ ID NO:246, lacking the signal peptide of SEQ ID NO:
 1. 65. The CoStAR of claim44, wherein the CoStAR comprises the amino acid sequence of SEQ ID NO:264, lacking the signal peptide of SEQ ID NO:
 1. 66. The CoStAR of claim44, wherein the CoStAR comprises the amino acid sequence of SEQ ID NO:282, lacking the signal peptide of SEQ ID NO:
 1. 67. The CoStAR of claim44, wherein the extracellular binding domain is connected to a linkerhaving an amino acid sequence of SEQ ID NO: 18, which is connected to aan amino acid sequence of SEQ ID NO: 19 that comprises a spacer sequenceand a transmembrane sequence, that is in turn connected to the CD28signaling domain, that is in turn connected to the CD40 signalingdomain.
 68. A cell which expresses a CoStAR, the CoStAR comprising: anextracellular binding domain that binds to mesothelin (MSLN); a CD28signaling domain, wherein the CD28 signaling domain comprises the aminoacid sequence of SEQ ID NO: 25; and a CD40 signaling domain, wherein theCD40 signaling domain comprises the amino acid sequence of SEQ ID NO:32, wherein the extracellular binding domain comprises any one of: (a) aHCDR1 that is an HCDR1 in SEQ ID NO: 186, a HCDR2 that is an HCDR2 inSEQ ID NO: 186, a HCDR3 that is an HCDR3 in SEQ ID NO: 186, a LCDR1 thatis an LCDR1 in SEQ ID NO: 186, a LCDR2 that is an LCDR2 in SEQ ID NO:186, and LCDR3 that is an LCDR3 in SEQ ID NO: 186; (b) a HCDR1 that isan HCDR1 in SEQ ID NO: 187, a HCDR2 that is an HCDR2 in SEQ ID NO: 187,a HCDR3 that is an HCDR3 in SEQ ID NO: 187, a LCDR1 that is an LCDR1 inSEQ ID NO: 187, a LCDR2 that is an LCDR2 in SEQ ID NO: 187, and LCDR3that is an LCDR3 in SEQ ID NO: 187; (c) a HCDR1 that is an HCDR1 in SEQID NO: 188, a HCDR2 that is an HCDR2 in SEQ ID NO: 188, a HCDR3 that isan HCDR3 in SEQ ID NO: 188, a LCDR1 that is an LCDR1 in SEQ ID NO: 188,a LCDR2 that is an LCDR2 in SEQ ID NO: 188, and LCDR3 that is an LCDR3in SEQ ID NO: 188; (d) a HCDR1 that is an HCDR1 in SEQ ID NO: 189, aHCDR2 that is an HCDR2 in SEQ ID NO: 189, a HCDR3 that is an HCDR3 inSEQ ID NO: 189, a LCDR1 that is an LCDR1 in SEQ ID NO: 189, a LCDR2 thatis an LCDR2 in SEQ ID NO: 189, and LCDR3 that is an LCDR3 in SEQ ID NO:189; (e) a HCDR1 that is an HCDR1 in SEQ ID NO: 190, a HCDR2 that is anHCDR2 in SEQ ID NO: 190, a HCDR3 that is an HCDR3 in SEQ ID NO: 190, aLCDR1 that is an LCDR1 in SEQ ID NO: 190, a LCDR2 that is an LCDR2 inSEQ ID NO: 190, and LCDR3 that is an LCDR3 in SEQ ID NO: 190; (f) aHCDR1 that is an HCDR1 in SEQ ID NO: 191, a HCDR2 that is an HCDR2 inSEQ ID NO: 191, a HCDR3 that is an HCDR3 in SEQ ID NO: 191, a LCDR1 thatis an LCDR1 in SEQ ID NO: 191, a LCDR2 that is an LCDR2 in SEQ ID NO:191, and LCDR3 that is an LCDR3 in SEQ ID NO: 191; (g) a VH that is a VHin SEQ ID NO: 186, and a VL that is a VL in SEQ ID NO: 186; (h) a VHthat is a VH in SEQ ID NO: 187, and a VL that is a VL in SEQ ID NO: 187;(i) a VH that is a VH in SEQ ID NO: 188, and a VL that is a VL in SEQ IDNO: 188; (j) a VH that is a VH in SEQ ID NO: 189, and a VL that is a VLin SEQ ID NO: 189; (k) a VH that is a VH in SEQ ID NO: 190, and a VLthat is a VL in SEQ ID NO: 190; or (l) a VH that is a VH in SEQ ID NO:191, and a VL that is a VL in SEQ ID NO:
 191. 69. The cell of claim 68,wherein the cell comprises an alpha-beta T cell, gamma-delta T cell, Tregulatory cell, TIL, NKT cell or NK cell.
 70. The cell of claim 68,wherein the cell coexpresses a CAR or a TCR.
 71. A fusion protein,wherein the fusion protein comprises: i. a first sequence, wherein thefirst sequence comprises one or more of: a. a HCDR1 that is an HCDR1 inSEQ ID NO: 186, a HCDR2 that is an HCDR2 in SEQ ID NO: 186, a HCDR3 thatis an HCDR3 in SEQ ID NO: 186, a LCDR1 that is an LCDR1 in SEQ ID NO:186, a LCDR2 that is an LCDR2 in SEQ ID NO: 186, and LCDR3 that is anLCDR3 in SEQ ID NO: 186; b. a HCDR1 that is an HCDR1 in SEQ ID NO: 187,a HCDR2 that is an HCDR2 in SEQ ID NO: 187, a HCDR3 that is an HCDR3 inSEQ ID NO: 187, a LCDR1 that is an LCDR1 in SEQ ID NO: 187, a LCDR2 thatis an LCDR2 in SEQ ID NO: 187, and LCDR3 that is an LCDR3 in SEQ ID NO:187; c. a HCDR1 that is an HCDR1 in SEQ ID NO: 188, a HCDR2 that is anHCDR2 in SEQ ID NO: 188, a HCDR3 that is an HCDR3 in SEQ ID NO: 188, aLCDR1 that is an LCDR1 in SEQ ID NO: 188, a LCDR2 that is an LCDR2 inSEQ ID NO: 188, and LCDR3 that is an LCDR3 in SEQ ID NO: 188; d. a HCDR1that is an HCDR1 in SEQ ID NO: 189, a HCDR2 that is an HCDR2 in SEQ IDNO: 189, a HCDR3 that is an HCDR3 in SEQ ID NO: 189, a LCDR1 that is anLCDR1 in SEQ ID NO: 189, a LCDR2 that is an LCDR2 in SEQ ID NO: 189, andLCDR3 that is an LCDR3 in SEQ ID NO: 189; e. a HCDR1 that is an HCDR1 inSEQ ID NO: 190, a HCDR2 that is an HCDR2 in SEQ ID NO: 190, a HCDR3 thatis an HCDR3 in SEQ ID NO: 190, a LCDR1 that is an LCDR1 in SEQ ID NO:190, a LCDR2 that is an LCDR2 in SEQ ID NO: 190, and LCDR3 that is anLCDR3 in SEQ ID NO: 190; f. a HCDR1 that is an HCDR1 in SEQ ID NO: 191,a HCDR2 that is an HCDR2 in SEQ ID NO: 191, a HCDR3 that is an HCDR3 inSEQ ID NO: 191, a LCDR1 that is an LCDR1 in SEQ ID NO: 191, a LCDR2 thatis an LCDR2 in SEQ ID NO: 191, and LCDR3 that is an LCDR3 in SEQ ID NO:191; g. a VH that is a VH in SEQ ID NO: 186, and a VL that is a VL inSEQ ID NO: 186; h. a VH that is a VH in SEQ ID NO: 187, and a VL that isa VL in SEQ ID NO: 187; i. a VH that is a VH in SEQ ID NO: 188, and a VLthat is a VL in SEQ ID NO: 188; j. a VH that is a VH in SEQ ID NO: 189,and a VL that is a VL in SEQ ID NO: 189; k. a VH that is a VH in SEQ IDNO: 190, and a VL that is a VL in SEQ ID NO: 190; or (a) a VH that is aVH in SEQ ID NO: 191, and a VL that is a VL in SEQ ID NO: 191; ii. asecond sequence that is a transmembrane domain; iii. a third sequencethat comprises the amino acid sequence of SEQ ID NO: 25; and iv. afourth sequence that comprises the amino acid sequence of SEQ ID NO: 32,wherein the first sequence is linked to the second sequence, wherein thesecond sequence is linked to the third sequence, and wherein the thirdsequence is linked to the fourth sequence.
 72. A method of cell therapycomprising: a) identifying a subject, wherein the subject has cancerthat expresses MSLN; and b) administering any a CoStAR that comprises:an extracellular binding domain that binds to mesothelin (MSLN), atransmembrane domain, a CD28 signaling domain that comprises the aminoacid sequence of SEQ ID NO: 25; and a CD40 signaling domain, wherein theCD40 signaling domain comprises the amino acid sequence of SEQ ID NO:32, wherein the extracellular binding domain is linked to thetransmembrane domain, wherein the transmembrane domain is linked to theCD28 signaling domain, wherein the CD28 signaling domain is linked tothe CD40 domain, and wherein the extracellular binding domain comprisesany one or more of: (a) a HCDR1 that is an HCDR1 in SEQ ID NO: 186, aHCDR2 that is an HCDR2 in SEQ ID NO: 186, a HCDR3 that is an HCDR3 inSEQ ID NO: 186, a LCDR1 that is an LCDR1 in SEQ ID NO: 186, a LCDR2 thatis an LCDR2 in SEQ ID NO: 186, and LCDR3 that is an LCDR3 in SEQ ID NO:186; (b) a HCDR1 that is an HCDR1 in SEQ ID NO: 187, a HCDR2 that is anHCDR2 in SEQ ID NO: 187, a HCDR3 that is an HCDR3 in SEQ ID NO: 187, aLCDR1 that is an LCDR1 in SEQ ID NO: 187, a LCDR2 that is an LCDR2 inSEQ ID NO: 187, and LCDR3 that is an LCDR3 in SEQ ID NO: 187; (c) aHCDR1 that is an HCDR1 in SEQ ID NO: 188, a HCDR2 that is an HCDR2 inSEQ ID NO: 188, a HCDR3 that is an HCDR3 in SEQ ID NO: 188, a LCDR1 thatis an LCDR1 in SEQ ID NO: 188, a LCDR2 that is an LCDR2 in SEQ ID NO:188, and LCDR3 that is an LCDR3 in SEQ ID NO: 188; (d) a HCDR1 that isan HCDR1 in SEQ ID NO: 189, a HCDR2 that is an HCDR2 in SEQ ID NO: 189,a HCDR3 that is an HCDR3 in SEQ ID NO: 189, a LCDR1 that is an LCDR1 inSEQ ID NO: 189, a LCDR2 that is an LCDR2 in SEQ ID NO: 189, and LCDR3that is an LCDR3 in SEQ ID NO: 189; (e) a HCDR1 that is an HCDR1 in SEQID NO: 190, a HCDR2 that is an HCDR2 in SEQ ID NO: 190, a HCDR3 that isan HCDR3 in SEQ ID NO: 190, a LCDR1 that is an LCDR1 in SEQ ID NO: 190,a LCDR2 that is an LCDR2 in SEQ ID NO: 190, and LCDR3 that is an LCDR3in SEQ ID NO: 190; (f) a HCDR1 that is an HCDR1 in SEQ ID NO: 191, aHCDR2 that is an HCDR2 in SEQ ID NO: 191, a HCDR3 that is an HCDR3 inSEQ ID NO: 191, a LCDR1 that is an LCDR1 in SEQ ID NO: 191, a LCDR2 thatis an LCDR2 in SEQ ID NO: 191, and LCDR3 that is an LCDR3 in SEQ ID NO:191; (g) a VH that is a VH in SEQ ID NO: 186, and a VL that is a VL inSEQ ID NO: 186; (h) a VH that is a VH in SEQ ID NO: 187, and a VL thatis a VL in SEQ ID NO: 187; (i) a VH that is a VH in SEQ ID NO: 188, anda VL that is a VL in SEQ ID NO: 188; (j) a VH that is a VH in SEQ ID NO:189, and a VL that is a VL in SEQ ID NO: 189; (k) a VH that is a VH inSEQ ID NO: 190, and a VL that is a VL in SEQ ID NO: 190; or (1) a VHthat is a VH in SEQ ID NO: 191, and a VL that is a VL in SEQ ID NO: 191.